Identifying root-associated bacteria and potential mechanisms linked to compost suppressiveness towards Fusarium oxysporum.

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Hildah Amutuhaire, Adi Faigenboim-Doron, Jonathan Friedman, Eddie Cytryn This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5349913/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 16 May, 2025 Read the published version in Environmental Microbiome → Version 1 posted 16 You are reading this latest preprint version Abstract Background Soilborne fungal phytopathogens pose a significant threat to global food security. While chemical control remains an effective method for managing these pathogens, increasing regulations due to health and environmental concerns, along with rising fungicide resistance, have restricted their use, underscoring the urgent need for sustainable alternatives. The use of compost to enhance soil fertility and suppress plant diseases is well documented. Several studies have underlined the role of microorganisms in disease suppression, but the mechanisms facilitating this disease suppression remain unclear. We evaluated the impact of compost amendment on the composition and functional capacity of the rhizosphere microbiome in cucumber plants ( Cucumis sativus ) inoculated with Fusarium oxysporum f. sp. radicis-cucumerinum (FORC) under controlled greenhouse conditions using amplicon sequencing, shotgun metagenomic and culture-based techniques. Results Compost amendment significantly reduced FORC-induced disease in cucumber relative to non-amended treatments. While FORC inoculation resulted in significant shifts in microbial (bacterial and fungal) community composition in the rhizosphere of non-amended plant, this phenomenon was substantially less pronounced in the rhizosphere of compost-amended plants. Specifically, compost amendment sustained the presence of Actinomycetota ( Streptomyces , Actinomadura , Saccharomonospora , Pseudonocardia , Glycomyces , Thermobifida ) and Bacillota ( Planifilum , Novibacillus ) in FORC inoculated plants, that diminished significantly in inoculated plants without compost. These taxa contained a myriad of non-ribosomal peptides (NRPS) and polyketides (PKS) biosynthetic gene clusters (BGCs) with putative antimicrobial and iron-chelating functions. We successfully isolated two Streptomyces strains from disease suppressed compost amended rhizosphere (almost identical to the most prominent strain identified in the molecular analyses) that produced extracellular metabolites that inhibited growth of FORC in-vitro. Genome analysis of these strains revealed BGCs that encode for compounds with potential antimicrobial capacity. Conclusions Based on results presented in this study, we demonstrate that compost alleviates FORC-induced dysbiosis of the rhizosphere microbiome, maintaining abundance of specific bacterial taxa. These bacterial groups may contribute to disease suppression through a myriad of mechanisms including iron chelation and production of fungal antagonizing secondary metabolites. Rhizosphere microbiome FORC compost suppressiveness NRPS PKS amplicon sequencing shotgun metagenomics Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Background Soilborne fungal phytopathogens pose a significant threat to global food security because they infect a wide range of crops and are difficult to control [ 1 , 2 ] especially since most of them can survive in soil in the absence of host plants [ 3 , 4 ]. The Fusarium oxysporum species complex includes several strains that cause wilt or root and crown rot in a variety of commercially significant crops [ 5 ]. These pathogens are particularly challenging for plant protection management [ 6 ] due to growing resistance of some Fusarium species to commonly used fungicides, such as azoles [ 7 ]. Chemical fungicides are often the most effective way to control soil borne fungal pathogens [ 4 ], but increased regulation due to human and environmental health ramifications has strongly restricted use of several conventionally applied compounds [ 8 ]. This phenomenon, coupled to increasing global demand for food production and the predicted geographic expansion of phytopathogens due to climate change, has created a dire need for sustainable alternatives [ 9 ]. Soil amendments, including animal and green manure, organic wastes [ 1 , 10 , 11 ], compost, [ 12 , 13 ] and more recently, biochar [ 14 , 15 ] are considered to be beneficial strategies for controlling soilborne pathogens. The capacity of compost to effectively suppress soilborne fungal pathogens has been previously demonstrated in a diverse array of studies [ 1 , 12 , 16 , 17 ] and while abiotic factors such as the presence of compost-derived fungitoxic chemicals have been reported, collective evaluation of current research suggests that the primary mechanisms behind compost disease suppressiveness are linked to shifts in the overall soil microbial community composition, diversity, activity and functioning [ 17 , 18 ], that facilitate enrichment of specific groups of bacteria with antagonistic activity against soilborne pathogens [ 19 , 20 ]. The rhizosphere microbiome enhances plant nutrient uptake, facilitates protection against abiotic stress, and acts as the first line of defense against soilborne fungal pathogens [ 21 ]. Certain rhizosphere bacteria protect plant roots from colonization by soilborne pathogens through a range of mechanisms, including competition for space and nutrients, parasitism, induction of systemic resistance [ 22 – 24 ]. In addition, various root-associated bacteria produce an array of small molecules known as secondary metabolites (SMs) that play vital ecological roles in interactions with the local microbiome, plant roots and soilborne pathogens, and subsequently impact plant growth and health [ 25 , 26 ]. Non-ribosomal peptides (NRPs) and polyketides (PKs) are among the most prevalent classes of SMs across the three life domains [ 27 ]. These compounds include iron scavenging siderophores, stress protectant pigments, signaling molecules, and antimicrobials, including antifungals [ 28 ]. NRPs and PKs are synthesized by non-ribosomal peptide synthetases (NRPS) and polyketide synthases (PKS) respectively [ 29 , 30 ], huge enzymatic complexes (typically more than 500 KDa) that are encoded by large biosynthetic gene clusters (BGCs). These complexes have a similar multi-modular architecture, consisting of repeating domains that are each in charge of incorporating a single building block into an expanding molecule in a stepwise fashion. The ability of NRPS and PKS to incorporate a wide range of building blocks enables them to assemble and customize a diverse array of chemical compounds with a variety of properties and functions [ 31 , 32 ], making them an untapped source of potentially novel antifungal compounds for application in agriculture as biofungicides. While numerous studies have demonstrated compost-facilitated disease suppressiveness in various pathosystems and identified bacterial taxa that are stimulated by compost amendment [ 19 , 33 ], few have deliberated the mechanisms responsible for disease suppression by compost-induced bacteria [ 34 ]. We hypothesize that compost enhances disease suppression by stimulating rhizosphere bacteria that antagonize pathogens at least partially through the production of antimicrobial secondary metabolites. This study aimed to identify microbial populations and associated secondary metabolite encoding genes involved in compost-induced soilborne pathogen suppression, using a model comprised of greenhouse grown cucumber plants inoculated with F. oxysporum f. sp. radicis-cucumerinum (FORC). Initially, we evaluated the impact of compost amendment on the microbial community composition of cucumber rhizospheres (with and without FORC inoculation) using 16S rRNA gene amplicon sequencing and identified specific bacterial populations that were potentially associated with the observed compost-induced suppressiveness. Subsequently, we identified functional genes potentially linked to compost facilitated suppressiveness using shotgun metagenomics, specifically focusing on genes encoding for secondary metabolites from compost stimulated bacterial taxa. Using culturomics, we isolated representative bacteria associated with compost-suppressiveness and tested their capacity to secrete FORC-antagonizing extracellular metabolites in-vitro. Materials and methods Greenhouse experiment For compost amended treatments, soil, commercial perlite (Perlite 212 Agrekal, Israel), and compost (Azur nurseries, Israel) were mixed in a 2:2:1 (v/v) ratio, whereas in non-amended treatments, soil was mixed with perlite in a 1:1 (v/v) ration. These soil mixtures were watered every other day for a week before transplanting seedlings (see below). A clay-rich soil from Ahituv, Israel (32.389988, 34.990965) used exclusively for growing cucumbers in greenhouses was used in this study. It was air-dried at room temperature, and then ground and sieved through a 2 mm mesh to ensure uniform particle size. Cucumber ( Cucumis sativus ) seeds (Manny- Genesis seeds, lot # 10614C) were sown in seedling trays filled with perlite:vermiculite mixture (1:3, respectively) and the trays were incubated in a temperature-controlled growth chamber (28–30°C) for 7 days and were watered every other day with tap water to facilitate germination. Germinated seedlings were then transplanted into 2.5-inch square black nursery pots (one plant per pot) containing the potting mixtures described above, producing the four following treatments: (i) non-inoculated, non-amended (5 plants), (ii) non-inoculated, compost-amended (5 plants), (iii) FORC-inoculated, non-amended (6 plants), and (iv) FORC-inoculated, compost-amended (6 plants). The inoculated treatments contained 1% FORC (w/w), prepared according to the protocol previously described by [ 35 ]. All plants were fertigated with 500X diluted commercial greenhouse liquid fertilizer for 21 days. Rhizosphere samples were collected by gently shaking off excess soil from the roots and collecting the remaining soil that was firmly adhered to the root surface. Half of each soil was stored in sterile 50 ml polypropylene tubes at -80 o C for DNA extraction, whereas the other half was mixed in glycerol (final concentration of 50%) and stored at -80 o C for subsequent strain isolation described below. Disease phenotyping To determine the capacity of compost to induce disease suppression, disease incidence, stem height, shoot dry weight, and root fresh weight were measured at 21 days post-inoculation. Disease incidence was determined by counting the number of wilted plants, shoot dry weight (g) was measured following overnight drying (100 o C) of plant excised shoots, and root fresh weight by measuring the weight of roots (g) after collection of rhizosphere soil samples. Rhizosphere sampling, DNA extraction, and sequencing DNA was extracted from 0.25 g of rhizosphere material of all plants (N = 22) using the DNeasy PowerSoil Kit (Qiagen, USA), according to the manufacturer's protocol. For characterization of bacterial and fungal communities, we PCR amplified the V3-V4 region of the 16S rRNA gene (bacteria) and the nuclear ribosomal internal transcribed spacer (ITS, fungi), using the 341F/806R [ 36 ] and ITS1-F/ITS2R [ 37 ] primer sets, respectively. The primers were appended with CS1(forward), and CS2 (reverse) Illumina sequencing adapters and sequencing was conducted using a paired-end 2x300bp cycle run on an Illumina MiSeq sequencing system using MiseqV3 chemistry, at Rush University Genomics and Microbiome Core Facility (Chicago, USA). Shotgun metagenomic sequencing was conducted on 12 DNA samples from the FORC-inoculated treatments (6 non-amended and 6 compost-amended) using an Illumina NovaSeq S4 platform with 2x150bp paired-end reads, employing Illumina dsDNA shotgun library preparation. The sequencing was performed at Rush University Genomics Microbiome Core Facility. All datasets have been deposited in the NCBI Short Read Archive under Bioproject PRJNA1169419 . Bioinformatic analysis of generated amplicon sequences Bacterial 16S rRNA gene and fungal ITS amplicons were demultiplexed and analyzed using QIIME2 (v2019.07) [ 38 ], after applying the DADA2 denoising pipeline [ 39 ] ( Table S1 , Additional file 2 ). Amplicon Sequence Variants (ASVs) with a frequency of less than 3 were removed. Taxonomic assignment of the ASVs was done using the QIIME2 q2-feature-classifier with the Silva 138 [ 40 ] and UNITE [ 41 ] databases, for bacterial and fungal ASV characterizations, respectively. A mid-point rooted phylogenetic tree for the calculation of diversity matrices was created with fasttree and mafft alignment (QIIME2) using the generated ASVs. Raw count tables, ASV taxonomy, and the rooted phylogenetic tree generated in QIIME2 were exported for downstream analyses and visualization in R (version 4.3.1). Quality control and assembly of shotgun metagenomic reads and characterization of assembled contigs The quality of raw shotgun metagenomic reads was assessed using FASTQC, and low-quality reads and adapters were removed using Trimmomatic [ 42 ] with default parameters for adapter trimming, and CROP:145 HEADCROP:15 SLIDINGWINDOW:4:20 MINLEN:100 parameters for low quality trimming. Subsequently, the reads were aligned against the FORC reference genome (GenBank GCA_001702695.2) using Bowtie2, --very-sensitive-local flag [ 43 ] and FORC reads were removed using SAMtools [ 44 ]. High quality FORC-filtered reads from all samples were pooled and assembled using MEGAHIT [ 45 ] ( Table S2 , Additional file 2 ). The assembled contigs were filtered to retain contigs > 2000bp, and these were used as input for prediction of open reading frames (ORFs) using Prodigal [ 46 ], in sensitive mode. The predicted ORFs were aligned to the NCBI-nr protein database using DIAMOND [ 47 ], and MEGAN V6 Ultimate Edition [ 48 ] was then used for taxonomic assignment of these ORFs based on the Lowest common ancestor (LCA). Detection and annotation of biosynthetic gene clusters (BGCs) from assembled shotgun metagenomic contigs Assembled contigs from compost-amended and non-amended FORC-inoculated treatments were filtered to maintain contigs that were > 5000bp length, which were used to identify predicted secondary metabolite encoding BGCs with antiSMASH v6 [ 49 ], using the following parameters: –cb-general –cb-subclusters –pfam2go –strictness relaxed –genefinding-tool prodigal-m mode. A biom table of reads mapping to BGC core regions was generated using BiG-MAP [ 50 ], and differential abundance analysis of compost amended and nonamended groups was performed using Maaslin2 [ 51 ] using Total Sum Scaling (TSS) normalized table. The taxonomy of the identified BGCs was assigned by aligning contigs to the NCBI nonredundant database using DIAMOND and LCA classification with MEGAN [ 48 ]. NRPS and PKS BGCs were aligned against the MiBiG 3.0 [ 52 ] database to predict their putative functions using DIAMOND [ 47 ] (cutoffs: e-value 50%), as previously described [ 14 ]. MAG binning and annotation Contigs were binned using metaWRAP v1.3.2 [ 53 ] with a minimum completeness of 75% and a maximum contamination of 5%. Binning was performed using MaxBin2 [ 54 ], metaBAT2 [ 55 ], and CONCOCT [ 56 ]. The resulting bins were dereplicated using dRep [ 57 ] v3.4.2 with default parameters. Taxonomic classification was achieved using the GTDB-Toolkit [ 58 ] v2.3.2 (GTDB-Tk) classify workflow. Statistical analysis Statistical differences in shoot dry weight and root fresh weight between treatments were evaluated using one-way ANOVA and multiple comparisons of means using the Tukey-Kramer (HSD) test (α = 0.05) in JMP software (JMP®, Version 17. SAS Institute Inc., Cary, NC, 1989–2023). Shannon, OTU richness and Bray-Curtis diversity indices (for evaluating alpha- and beta-diversity of bacterial and fungal communities, respectively) were calculated using the Vegan R package. Differential abundance analysis of amplicon-generated count tables was performed using MaASlin2 [ 51 ] and Lefse [ 59 ]. Bacteria isolation, identification and whole-genome sequencing Selected bacteria were isolated from glycerol stocks of compost-induced disease suppressed rhizosphere samples using ccucumber plant-extract media prepared according to [ 60 ] with several modifications. Briefly, 20g of cucumber shoots were washed in tap water to remove any soil particles, cut into small pieces, and blended with minimum amount of water in a waring blender. The resulting extract was filtered through a triple layered gauze and stored at 4 o C as a stock solution. Agar plates were prepared by diluting the cucumber extract with distilled water in a ratio of 1:10 (v/v) and addition of 1.5% (w/v) Bacto agar (Difco Laboratories, Detroit). The medium was sterilized by autoclaving at 121 o C for 30 minutes and 50 mg/l of cycloheximide was added to preclude the growth of fungi. Cultures were incubated for 14 days at 30 o C, and the isolated bacteria were taxonomically identified based on 16S rRNA gene amplicon sequencing using primers; 11F (GTTTGATCCTGGCTCAG) and 1392R (ACGGGCGGTGTGTRC) [ 61 ]. The PCR products were sequenced by Macrogen Inc. (Seoul, Korea) and aligned against the NCBI nr database using BLASTn. High molecular weight genomic DNA was isolated from Streptomyces CC6 and CC27 isolates using MagAttract HMW DNA (Qiagen, USA) extraction Kit following the manufacturer’s protocol. Hybrid whole genome sequencing and assembly were performed by Plasmidsaurus Inc. (USA) service using Oxford Nanopore and Illumina Sequencing Technologies. Screening for in-vitro antifungal activity. Green fluorescent protein-labeled (gfp) FORC [ 62 ] was exposed to cell-free supernatants (CFS) from Streptomyces CC6 and CC27 to assess potential presence of inhibitory secreted metabolites, using the methodology developed by [ 63 ]. Briefly, CFS were prepared from bacterial spent media as follows; one milliliter of 3 days old starter culture was used to inoculate 50 ml of Tryptic Soy Broth (TSB, BD Bacto™ Soybean-Casein Digest Medium) in 250ml Erlenmeyer flasks. The flasks were incubated for 7 days at 30 o C and 170 rpm. The bacterial spent media was then centrifuged (5000g for 10 minutes) and the supernatant was filtered using a 0.22µm Millex-HV syringe filter (Sigma-Aldrich, Israel). Spore solution was prepared by inoculating a plug of gfp-labeled FORC mycelia taken from a 5 days old PDA (potato dextrose agar) plate in 25ml of fresh PDB (potato dextrose broth) media in 250ml Erlenmeyer flasks with and flasks were incubated for 7 days 25 o C and shaking at 100 rpm. FORC culture was filtered through 40 µm filters, and concentration was determined by counting spores using a Hemocytometer. In-vitro antifungal assays were carried out in 96-microwell plates by adding 100µl of bacterial cell-free supernatant (CFS) into 100µl of gfp-labelled FORC spores (10 5 spores/ml). CFS-treated FORC spores were incubated at 25°C for 24 h, and growth changes were monitored by measuring fluorescence and OD using the multimode Microplate Reader (Synergy H1, BioTek, USA). For dual culture plate assay, Streptomyces CC6 and CC27 starter cultures were grown on LB medium (Luria-Bertani) for 3 days. Ten microliters of the bacteria culture were spot inoculated in quadruplets on half strength LB agar plates, 2cm from the center of the plate. Plates were incubated at 30 o C for 5 days and thereafter a plug of 7 days old FORC mycelium was placed in the center of plate. The plates co-inoculated with bacterial and fungi were incubated at 28 o C and visually monitored for a period of 7 days. Results Compost amendment alleviates FORC-induced disease in cucumbers Disease incidence in FORC-inoculated cucumber plants was determined by counting the number of wilted plants in soil with and without compost at 21 days post inoculation. No mortality was recorded for inoculated plants grown in compost-amended soil, while 50% mortality (N = 6) was observed in inoculated plants grown in non-amended soil (Fig. 1 ). Additionally, FORC inoculation significantly reduced shoot dry weight and root fresh weight of inoculated cucumber plants grown in compost-free soil (Tukey HSD test, P 0.05) ( Fig. 1 ). Compost amendment reduces the relative abundance of Fusarium, and alleviates FORC-induced reduction in rhizosphere microbial diversity and shifts in community composition We evaluated the impact of compost amendment on the rhizosphere bacterial and fungal diversity and community composition in FORC-inoculated and non-inoculated plants using fungal ITS and bacterial 16S rRNA gene amplicon sequencing. We did not observe significant differences in bacterial community richness and phylogenetic diversity ( P > 0.05, Tukey HSD; Figure S1 A and S1B, Additional file 1 ) across all groups (with and without compost, in both the presence and absence of FORC). However, FORC inoculation significantly decreased bacterial species evenness (by 2.7%; Pielou's evenness index) compared to non-inoculated plants without compost ( P < 0.05, Tukey HSD). Pathogen inoculation caused a 1.56% reduction in the bacterial species evenness in compost amended plants when compared to non-inoculated plants without compost, however this was not statistically significant, P > 0.05, Tukey HSD (Fig. 2 A, and Figure S1 C, Pielou's evenness index, Additional file 1 ). Without FORC, compost amendment slightly reduced fungal diversity in the rhizosphere. However, without compost FORC inoculation facilitated a 32% ( P < 0.05, Tukey HSD) decrease in fungal diversity, whereas in compost amended samples the decrease was only 13% and was not significant compared to the compost-amended samples not inoculated with FORC (Fig. 2 B, and Figure S2 A, Additional file 1 ). Distinct bacterial and fungal communities were observed in compost amended vs. non-amended cucumber plants (Bray-Curtis Dissimilarity matrix), and the amendment group explained 59% and 53.6% (Adonis test - amendment group R 2 = 0.59 and 0.536, bacterial 16S rRNA and fungal ITS respectively, P < 0.05) of the differences in bacterial and fungal communities respectively (Fig. 2 C and 2 D). In the absence of compost, bacterial and fungal communities were significantly affected by FORC inoculation, as opposed to the compost amended communities in which FORC-induced shifts were considerably less pronounced. Estimation of the relative abundance of FORC-associated ITS amplicon sequence variants (ASV) and of FORC-characterized reads from shotgun metagenomic analyses, indicated that compost amendment significantly reduced the relative abundance of FORC in the rhizosphere ( Figure S2 B, S2C and S2D, Additional file 1 ). In summary, compost significantly reduced fungal diversity but did not have notable impact on bacterial diversity. It significantly altered the composition of both bacterial and fungal communities in addition to mitigating the negative impact of FORC inoculation by reducing FORC abundance and minimizing microbial community shifts in the rhizospheres of FORC inoculated cucumber plants. Considering the trends described above, previous reports suggesting bacteria are involved in suppressiveness and the vast reservoir of unexplored antifungal compounds produced by rhizosphere bacteria, we chose to hereafter focus on the bacterial community. Compost amendment mitigates FORC-induced reduction in the relative abundance of the Actinomycetota and Bacillota To identify bacterial populations potentially involved in facilitating compost suppressiveness, we taxonomically annotated the rhizosphere communities in all treatments and performed differential abundance analysis on the 16S rRNA gene amplicon sequencing data using LEfSe. FORC inoculation significantly reduced the relative abundance of Bacillota and Actinomycetota in the absence of compost amendment, but this reduction was not significant in the compost-amended treatments. Specifically, in FORC inoculated cucumber plants the relative abundance of the Bacillota Thermoactinomycetaceae , Clostridiales Family_XI, and Peptococcaceae , as well as Actinomycetota families Streptomycetaceae , Thermomonosporaceae , Glycomycetaceae , Propionibacteriaceae , Nocardiopsaceae , Intrasporangiaceae , Pseudonorcadiaceae and unclassified Micrococcales families was significantly higher in the rhizosphere of the compost-amended treatments than in the plants that were not amended with compost ( Fig. 3 ) . To validate 16S rRNA gene amplicon results and achieve high-resolution taxonomic classification, we performed shotgun metagenomics on FORC-inoculated samples (with and without compost), binning 116 medium- to high-quality dereplicated MAGs (> 70% completion, < 5% contamination) using the metaWRAP pipeline. Taxonomic classification (using the GTDB_tk classify workflow) revealed that MAGs from Actinomycetota and Bacillota were significantly associated with compost amendment ( Table S5, Additional file 2 and Figure S3, Additional file 1 ). These included genera such as Streptomyces , Actinomadura , Saccharomonospora , Pseudonocardia , Glycomyces , Thermobifida , Planifilum , and Novibacillus , ( Figure S4, Additional file 1 ), consistent with the compost-associated families identified in the 16S rRNA gene amplicon analysis. Collectively, these results suggest that compost amendment mitigated FORC-induced dysbiosis of the rhizosphere microbiome, maintaining high relative abundance of specific Bacillota and Actinomycetota genera. Actinomycetota and Bacillota associated SM-encoding gene clusters are highly abundant in disease suppressed compost-amended cucumber rhizospheres. We postulated that the compost-facilitated suppressiveness and alleviation of FORC-induced dysbiosis, was at least partially linked to bacterial secondary metabolites that antagonize FORC. To identify secondary metabolite-encoding gene clusters, contigs larger than 5000bp were pooled and used as input for antiSMASH v6 and BiG-MAP tool was used to generate gene cluster abundance table, focusing only on the core genes of a BGC. In total, 1237 BGCs were identified in FORC inoculated samples with and without compost ( Table S3, Additional file 2 ). While no differences in the BGCs diversity (Shannon index: Figure S5A, Additional file 1 ) were observed between compost amended and non-amended samples, there was a significant difference in the BGC composition between these two groups (Fig. 4 A). BGCs phylogenetically linked to Actinomycetota and Bacillota were significantly more abundant in disease suppressed compost amended cucumber rhizospheres, while Pseudomonadota and Bacteroidota BGCs were more prevalent in non-amended FORC-inoculated rhizospheres (Figure S5B, Additional file 1 ). Compost-enriched BGCs were mainly taxonomically associated with the Actinomycetota families Streptomycetaceae, Thermomonosporaceae, Pseudonorcadiaceae, Glycomycetaceae , and Nocardiopsaceae , and the Bacillota family Thermoactinomycetaceae , corresponding to the results of 16S rRNA gene amplicon analysis ( Fig. 4 B ). The compost “protected” bacterial families harbor diverse classes of SM-encoding BGCs including NRPS, PKS (T1, T2 and T3), siderophores, betalacones, lanthipeptides (classes i-iii), ectoine and terpenes, with Streptomycetaceae harboring the most abundant and diverse collection of SMs ( Fig. 4 C ) . In summary, although the diversity of BGCs was similar in compost-amended and non-amended FORC-inoculated rhizospheres, their composition differed significantly. The rhizospheres of disease-suppressed, compost-amended plants were enriched in a variety of BGCs from the Actinomycetota and Bacillota families, including Streptomycetaceae , Thermomonosporaceae , Pseudonocardiaceae , Glycomycetaceae , Nocardiopsaceae , and Thermoactinomycetaceae , consistent with the results of 16S rRNA gene amplicon analysis. Compost stabilized bacteria possess NRPS and PKS BGCs encoding for antimicrobial compounds and siderophores NPRS and PKS secondary metabolites are known to play important ecological roles in microbe-microbe and microbe-plant interactions in soil and roots microbiomes. To shed light on the potential functions of the identified NRPS and PKS gene clusters in compost amended, FORC suppressive rhizospheres, we focused on NRPS and PKS BGCs from the Actinomycetota and Bacillota taxa, due to their profuseness in these samples. A total of 160 NRPS- and PKS-encoding BGCs were aligned against the MIBiG database using Diamond blastx and function was assigned if a BGC had an E-value 50% similarity to a documented secondary metabolite producing cluster [ 14 ]. In total, 118 out of the 160 identified NRPS/PKS BGCs displayed statistically significant differences (MaAsLin2, P adj < 0.05) in abundance between the compost-amended and non-amended treatments ( Table S4, Additional file 2) , 47% (56/118) of whom were functionally categorized. The functionally annotated NRPS and PKS BGCs were primarily phylogenetically associated with the Actinomycetota phylum, in contrast to those affiliated with Bacillota phylum (particularly; Thermoactinomycetaceae , unclassified Negativicutes and Bacillales ) that could not be assigned a functional role according to our cutoff criteria. The annotated NRPS and PKS BGCs were primarily linked to production of antimicrobial compounds and siderophores (Fig. 5 A and 5 B). The antimicrobial encoding NRPS and PKS BGCs in disease suppressed, compost amended samples were predominantly associated with the Streptomyces genus. These included NRPS BGCs encoding for atratumycin, enduracidin, cadaside A, and maduralactomycin A antimicrobials; and PKS BGCs encoding for concanamycin A, azalomycin F3a, A-47934, and diazepinomicin antimicrobials (Fig. 5 A and 5 B). Other notable antimicrobial encoding BGCs include: Actinomadura -associated NRPS and PKS BGCs encoding for coprisamide C and merochlorin A, respectively; the NRPS taromycin A gene cluster from Saccharomonospora ; PKS loseolamycin A1; and griseorhodin A from Glycomyces and Thermobifida . Siderophores were mainly encoded by NRPS BGCs (Fig. 5 A). Additionally, NRPS gene clusters encoding for siderophore production were found in compost-stimulated Saccharomonospora (mirubactin), Thermobifida (fuscachelin A), Pseudonocardiaceae (saccharochelin A and albachelin), and Actinomadura (saccharochelin A) families, but not in Streptomyces . Overall, NRPS and PKS BGCs enriched in compost-amended, FORC-inoculated rhizospheres were primarily associated with antimicrobial and siderophore production. Most antimicrobial BGCs were associated with the Streptomyces genus, and the majority of siderophore encoding BGCs belonged to the Saccharomonospora , Thermobifida , and Actinomadura genera. Compost stimulated Streptomyces excrete FORC-antagonizing metabolites The contig-based functional annotation of NRPS and PKS BGCs highlighted the Streptomycetaceae family as a potential producer of antimicrobial compounds in the compost-amended FORC inoculated samples. Using plant-based medium, we isolated two Streptomyces strains (CC6 and CC27) from compost induced, disease suppressed rhizosphere samples. 16S rRNA gene analysis confirmed that these strains clustered with the Streptomyces ASV_2 (Fig. 6 A), whose abundance was slightly augmented by compost in the presence of FORC, in contrast to plants not amended with compost where it was significantly lower in FORC inoculated samples ( Figure S6A, Additional file 1 ). Additionally, the genomes of these two isolates were over 99% identical to the Streptomyces bin2 MAG (ortho ANI) ( Table S6, Additional file 2 and Figure S6B, Additional file 1 ), confirming isolation of these key strains. The genomes of CC6, CC27, and bin2 share 11 unique BGCs not found in MIBiG, along with a transAT-PKS-NRPS cluster sharing 93% of its genes with the weishanmycin BGC and a HR-T2PKS-NRPS hybrid cluster sharing 55% of its genes with the ishigamide BGC ( Figure S7, Additional file 1 ). Streptomyces CC6 and CC27 extracts significantly inhibited growth ( in- vitro) of FORC (Fig. 6 B). The fungal inhibitory phenotype was validated using a bacteria-fungal dual culture plate assay (Fig. 6 C). Discussion The ability of compost to suppress soilborne pathogens in various pathosystems is well established [ 1 , 12 , 16 ]. Evidence indicates that while physicochemical properties of compost contribute to its suppressiveness, sterilization leads to a loss of this effect, underscoring the crucial role of microorganisms [ 13 , 17 , 64 ]. While various processes such as altering soil physicochemical characteristics to promote microbial growth and activity, and augmenting the native rhizosphere microbiome by introducing new microbes [ 11 , 65 , 66 ] have been associated with compost-induced suppressiveness, the specific microbial populations and mechanisms driving compost induced disease suppression remain unclear. This study aimed to identify key rhizosphere bacterial communities and genes related to secondary metabolite production, potentially involved in compost-mediated suppression of FORC in greenhouse grown cucumbers. Greenhouse experiments validated that compost amendment mitigated FORC-induced disease, and microbial analyses revealed that this phenomenon was linked to a significant reduction in the relative abundance of FORC in the rhizosphere. While compost amendment altered the rhizosphere microbial community composition when compared to non-amended samples, our findings indicate that it also "stabilized" the rhizosphere microbiome in the presence of FORC, mitigating pathogen-induced reduction of microbial diversity and alleviating shifts in community composition. Compost may stabilize the rhizosphere microbiome in the presence of a pathogen by altering the soil physicochemical characteristics such as pH and organic carbon to promote proliferation of the native microbiome or augmentation of the native microbial communities by introducing in new species [ 11 , 13 , 18 , 34 , 66 ]. We propose that compost may facilitate disease suppression by alleviating pathogen-induced dysbiosis of the rhizosphere microbiome, maintaining robustness of specific bacterial populations. Our findings are supported by previous studies that found that compost amendment led to sustained alterations in the structure of soil microbial communities [ 12 , 65 , 66 ], however these were all conducted without addition of pathogens. The compost stimulated eubiosis of the FORC-amended rhizosphere microbiome was most prominent in the case of selected Bacillota and Actinomycetota genera that were highly abundant in FORC inoculated plants with compost, but almost completely absent in the compost-free inoculated treatments. This is supported by past studies that found positive correlations between Actinomycetota and Bacillota abundance, and inhibition of soilborne pathogens such as Fusarium oxysporum [ 67 , 68 ] and Ralstonia solanacearum [ 69 ]. Interestingly, reduction of these phyla in tomato rhizosphere was associated with increased disease incidence [ 69 ]. Delving into the 16S rRNA and MAG binning of shotgun metagenomic data revealed significantly higher relative abundance of Streptomyces , Actinomadura , Saccharomonospora , Pseudonocardia , Glycomyces and Thermobifida ( Actinomycetota ) and of Planifilum and Novibacillus ( Bacillota ) in FORC-inoculated rhizosphere samples amended with compost, compared to inoculated plants without compost. These genera were enriched with genes encoding production of diverse secondary metabolites including NRPS and PKS genes associated with production of antimicrobial and siderophore compounds, suggesting that they may directly antagonize soilborne pathogens through excreted antimicrobial and iron scavenging compounds. Previous studies indicated that Actinomycetota and Bacillota strains can directly antagonize Fusarium and other pathogens through production of antibiotics and lytic enzymes, and indirectly by competition such as production of siderophores [ 70 , 71 ]. Secondary metabolites produced by non-ribosomal peptide synthetases (NRPS) and polyketide synthases (PKS) are known to play crucial roles in antagonizing fungal pathogens in the rhizosphere [ 14 , 25 ]. Streptomyces are known to produce a wide range of antimicrobial compounds and includes strains that have been shown to antagonize Fusarium wilts in several in-planta studies [ 68 , 72 ]. We found that Streptomyces spp. were associated with the majority of NRPS and PKS gene clusters predicted to produce antimicrobial compounds in the compost-amended FORC inoculated samples. We successfully isolated two Streptomyces strains from the compost-induced FORC-suppressing cucumber rhizospheres that secrete FORC-inhibiting extracellular metabolites in-vitro. Their genomes contained NRPS-transAT-PKS and HR-T2PKS-NRPS hybrid clusters that encoded for potential congeners of weishanmycin and ishigamide, respectively. Weishanmycin is a Streptomyces -produced congener of the well-known, highly potent antitumor drug leinamycin [ 73 ]. Nonetheless, it may also have antimicrobial attributes, however this along with its native ecological role is currently unknown. Ishigamide is a Streptomyces derived amide-containing polyene whose function is unknown [ 74 ]. Polyenes are broad-spectrum antifungal agents produced by Streptomyces that act by increasing the permeability of the fungal cell membrane through targeting ergosterol, resulting in fungal cell death [ 75 ]. We posit that these, and potentially additional Streptomyces -produced compounds could play a crucial role in compost-induced disease suppression through direct fungal antagonism. It is imperative to emphasize that our findings represent a specific compost in a specific pathosystem and may not be universal. Future studies should emphasize the potential role of fungi in compost-induced disease suppression and investigate the effects of different composts on rhizosphere microbial communities and functions, as well as their role in disease suppression across various pathosystems. Additionally, designing consortia of strains maintained in eubiosis by compost could help determine their ability to promote disease suppression under disease-conducive conditions. Beyond the production of antimicrobial secondary metabolites, other compost suppressive mechanisms, such as the production of fungal lytic enzymes and activation of plant-induced systemic resistance (ISR), also merit attention. Conclusions This study underlines a link between compost, eubiosis of the rhizosphere microbiome, and mitigation of soilborne pathogen facilitated plant disease, which appears to be analogous to prebiotic-facilitated eubiosis of the gut microbiome that prevents disease in mammals. We posit that compost facilitates a buffering effect that protects the rhizosphere microbiome from pathogen-induced dysbiosis, sustaining Actinomycetota and Bacillota associated strains that potentially antagonize Fusarium through a range of mechanisms including the production of antimicrobial secondary metabolites and siderophores. These findings can be exploited to promote sustainable crop protection strategies that preserve beneficial rhizosphere microbiomes, and to isolate pathogen-antagonizing strains (and their metabolites) that can be as sustainable alternatives to chemical pesticides. Declarations Acknowledgements We thank Elad Cohen for his support with computational resources. Authors’ contributions HA and EC conceptualized and designed the experiments. HA performed the experiments and data analyses. ADF performed shotgun metagenome assembly. HA, JF and EC wrote the manuscript. All authors contributed to the article and approved the submitted version. Funding This study was funded by Israel Ministry of Agriculture and Rural Development Chief Scientist grant 20-03-0061. Availability of data and materials The raw sequencing data have been deposited in the NCBI Sequence Read Archive (SRA) database under Bioproject PRJNA1169419. Declarations Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. Author details EC -Institute of Soil, Water and Environmental Sciences, Agricultural Research Organization, Rishon Lezion, Israel. JF -The Institute of Environmental Sciences, The Hebrew University of Jerusalem, Rehovot, Israel. HA - Institute of Soil, Water and Environmental Sciences, Volcani Institute, Agricultural Research Organization, Rishon-Lezion, Israel: Department of Plant Pathology and Microbiology, The Robert H. 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(B) relative abundance of fungal ITS ASVs taxonomically classified to genus level, (C) relative abundance of ITS ASV taxonomically classified as Fusarium genus and most abundant in FORC-inoculated samples with and without compost, (D) percentage of shotgun metagenomics reads mapped to the genome of F. oxysporum f. sp. radicis-cucumerinum (FORC). Figure S3. Phylogenetic tree and abundance of bins assembled from FORC-inoculated, compost amended and non-amended samples. Figure S4. Abundance of Actinomycetota and Bacillota associated MAGs assembled from FORC-inoculated, compost amended and non-amended samples and classified to genus level. Figure S5. Alpha diversity (observed and shannon) (A) of BGCs identified in the rhizosphere metagenomes of FORC-inoculated compost amended and non-amended plants. Phylum-level taxonomy of identified (by antiSMASH-V6.0) secondary metabolite-encoding biosynthetic gene clusters (BGCs) based on analysis of shotgun metagenome contigs (>5kbp) from compost-amended and non-amended FORC-inoculated cucumber rhizosphere samples (B). Figure S6. (A) Relative abundance of 16S rRNA gene amplicon ASVs classified as Streptomyces. Relative abundance is based on 16S rRNA gene amplicon count table for all samples. (B) Progressive Mauve alignment of the Streptomyces CC7 isolate genome and the Streptomyces bin2. Figure S7. BiG-SCAPE network of BGCs identified in Streptomyces isolates (CC6 and CC27) and bin2 genomes in comparison to the known BGCs in the MiBIG database. AmutuhaireetalAdditionalfile2.xlsx Additional file 2 Table S1. Summary of 16S rRNA and ITS amplicon sequencing reads. Table S2. Summary of shotgun metagenome generated reads and assembly. Table S3. List of all AntiSMASH identified BGCs within pooled samples compost amended and non-amended samples. Table S4. List of Actinomycetota and Bacillota NRPS and PKs BGCs annotated using MiBiG. Table S5. Summary statistics of all generated MAGs binned from compost amended and non-amended samples. Table S6. Ortho ANI values of Streptomyces MAG bin2 and isolates CC6 and CC27. 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Also discoverable on Platform About Our Team In Review Editorial Policies Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5349913","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":375451834,"identity":"db6f9d5f-15a6-4bc7-9b4a-8e17ca86ff15","order_by":0,"name":"Hildah Amutuhaire","email":"","orcid":"","institution":"Hebrew University of Jerusalem","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Hildah","middleName":"","lastName":"Amutuhaire","suffix":""},{"id":375451843,"identity":"3b530868-8bc1-41fe-b027-f1bb94d88022","order_by":1,"name":"Adi Faigenboim-Doron","email":"","orcid":"","institution":"Agricultural Research Organization","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Adi","middleName":"","lastName":"Faigenboim-Doron","suffix":""},{"id":375451848,"identity":"c714c278-172d-4bbf-aca0-55fe48396abe","order_by":2,"name":"Jonathan Friedman","email":"","orcid":"","institution":"Hebrew University of Jerusalem","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jonathan","middleName":"","lastName":"Friedman","suffix":""},{"id":375451849,"identity":"744a22dc-d98b-4c76-beda-638231730b02","order_by":3,"name":"Eddie Cytryn","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABCklEQVRIiWNgGAWjYFCCBIMDDBUMDHxgNohghojz8OHVcoaBgQ1DCxseLQyMbVAtKACXFn725I0HPs47LMfGfvjZgwcMdnbb2xmYX3xsY5DBpUWy51nBwZnbDhuz8aSZGyQwJCfPOczAZjmzDbfDDG7kGBzm3ZaW2CbBYCaRwMCcLMHMwGbMcwa3FnuQlr9zQFrYvwG11BPWYiAB1MLYYAPUwgOy5bAdUAvzY54K3FokzgD90nPMBuiXnDKJBIPjCRLMjG2MMyokcGrhb0/e/OFHjYQcP/vxbZI/KqrtJfgPH/7wwcDGnh+HFnR3MiQ2AKNJAmg9cRpAwB6ImT8Qr34UjIJRMApGAAAA1PVLSVqDiRgAAAAASUVORK5CYII=","orcid":"","institution":"Agricultural Research Organization","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Eddie","middleName":"","lastName":"Cytryn","suffix":""}],"badges":[],"createdAt":"2024-10-28 23:08:09","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5349913/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5349913/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s40793-025-00710-9","type":"published","date":"2025-05-16T15:57:07+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":68562990,"identity":"ce8b64ca-87c8-4704-8b9c-f696da738067","added_by":"auto","created_at":"2024-11-08 14:39:26","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":359376,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImpact of compost amendment on disease incidence and growth inhibition in FORC inoculated treatments. \u003c/strong\u003eRoot fresh weight (\u003cstrong\u003eA\u003c/strong\u003e) and shoot dry weight (\u003cstrong\u003eB\u003c/strong\u003e) of compost-amended and non-amended cucumber plants with and without pathogen inoculation\u003cstrong\u003e.\u003c/strong\u003e Different letters indicate statistically significant differences obtained by multiple comparison of all pairs of means (Tukey HSD test, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05). Disease incidence (\u003cstrong\u003eC\u003c/strong\u003e) and photographs (\u003cstrong\u003eD\u003c/strong\u003e) of compost amended and non-amended FORC-inoculated cucumber plants. Disease incidence was determined by calculating the percentage of wilted plants in each soil amendment at 21 days post inoculation. N = 6 and 5 for FORC-inoculated and non-inoculated respectively.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-5349913/v1/7563e9a8f3caf2d2f376dc48.png"},{"id":68562992,"identity":"4839daec-4a32-4206-af91-0983c9da7d36","added_by":"auto","created_at":"2024-11-08 14:39:26","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":113135,"visible":true,"origin":"","legend":"\u003cp\u003eDiversity\u003cstrong\u003e (\u003c/strong\u003eShannon diversity index\u003cstrong\u003e) \u003c/strong\u003eof\u003cstrong\u003e \u003c/strong\u003ebacterial \u003cstrong\u003e(A)\u003c/strong\u003e and fungal \u003cstrong\u003e(B)\u003c/strong\u003e communities in the rhizosphere of compost amended and non-amended plants with and without FORC inoculation. Different letters indicate statistically significant differences obtained by multiple comparison of all pairs of means (Tukey HSD test, \u003cem\u003eP\u003c/em\u003e\u0026nbsp;\u0026lt; 0.05). PCoA ordination based on Bray-Curtis dissimilarity matrices of bacterial 16S rRNA gene \u003cstrong\u003e(C)\u003c/strong\u003e and fungal ITS gene \u003cstrong\u003e(D) \u003c/strong\u003eamplicons\u003cstrong\u003e. \u003c/strong\u003eThe fungal ASV that was classified as \u003cem\u003eFusarium\u003c/em\u003e only present in FORC-inoculated samples, was excluded from this analysis. Compost amendment explained 59.2% and 52.7% of the differences in bacterial and fungal communities respectively (Adonis test - amendment group R\u003csup\u003e2 \u003c/sup\u003e= 0.59 and 0.57 respectively, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-5349913/v1/2ece474db8c079a1850e21d0.png"},{"id":68564061,"identity":"1859034f-8dcb-4542-b94d-df4d4a11aaaf","added_by":"auto","created_at":"2024-11-08 14:47:26","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":87950,"visible":true,"origin":"","legend":"\u003cp\u003eRelative abundances of bacterial phyla with significant differential abundance in rhizospheres of FORC-inoculated and non-inoculated and compost-amended and non-amended treatments (based on 16S rRNA gene ASVs)\u003cstrong\u003e (A)\u003c/strong\u003e. LDA scores of Actinomycetota \u003cstrong\u003e(B)\u003c/strong\u003e and Bacillota \u003cstrong\u003e(C)\u003c/strong\u003e families showing statistically significant differences between compost-amended (red) and nonamended (blue) FORC-inoculated cucumber plants, determined by LEfSe analysis. LDA score \u0026gt; 2.0 and FDR-adjusted P \u0026lt; 0.1.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-5349913/v1/261bd75a067933a4f8c59063.png"},{"id":68565502,"identity":"f05691d0-79fd-427b-b336-68c761f3eac7","added_by":"auto","created_at":"2024-11-08 14:55:26","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":210187,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e PCoA ordination of the identified BGCs based on Bray-Curtis dissimilarity matrices. The amendment factor explained 81% of the differences observed in BGC composition between compost amended and non-amended pathogen-inoculated plants (Adonis test: pairwise permanova - amendment group R2 = 0.81, P \u0026lt; 0.05). \u003cstrong\u003e(B)\u003c/strong\u003e Family level taxonomic classification of Actinomycetota and Bacillota BGCs, highly abundant in the rhizosphere of compost amended FORC inoculated plants (MaAsLin2, coef \u0026gt; 2, P adj \u0026lt; 0.05). + indicates compost stimulated families that were also identified in 16S rRNA gene amplicon sequencing analysis. \u003cstrong\u003e(C)\u003c/strong\u003e BGC classes of compost protected bacterial families that were more abundant in compost-amended samples. Singleton BGCs were grouped as others.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-5349913/v1/ce911e41c719cec13685e01c.png"},{"id":68564063,"identity":"f8dd1693-2777-4861-937d-0546df119bf4","added_by":"auto","created_at":"2024-11-08 14:47:26","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":275703,"visible":true,"origin":"","legend":"\u003cp\u003eDifferential abundance (normalized by z-score scaling of rows) of functionally characterized NRPS (\u003cstrong\u003eA), \u003c/strong\u003eand PKS (\u003cstrong\u003eB\u003c/strong\u003e) encoding BGCs in compost-amended vs. non-amended cucumber rhizospheres. Differential abundance analysis was conducted using MaASlin2, and functional annotation was based on \u0026gt;50% amino acid identity and E value of \u0026lt; 10\u003csup\u003e−40\u003c/sup\u003e of the identified NRPS/PKS BGCs to BGC sequences in MIBiG database. Taxonomic classification of BGCs was achieved using MEGAN V6 Ultimate Edition based on the Lowest common ancestor (LCA). The numbers in parentheses show the count of that specific BGC within the same genus, while the letters indicate that the BGC is taxonomically associated with different genera.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-5349913/v1/e25344bcefdbf73c202ddec9.png"},{"id":68562994,"identity":"b55ed013-20fd-4d47-bad9-f3623ea97694","added_by":"auto","created_at":"2024-11-08 14:39:26","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":392915,"visible":true,"origin":"","legend":"\u003cp\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Phylogenetic analysis of Streptomyces 16S rRNA gene amplicon ASVs and the isolates CC6 and CC27 Streptomyces 16S rRNA gene. Streptomyces CC6 and CC27 cluster together with Streptomyces ASV_2. MAFFT v6.864 online version was used for Multiple sequence alignment and construction of UPGMA (Unweighted Pair Group Method with Arithmetic Mean) phylogenetic tree and the tree and visualized using iTOL. (\u003cstrong\u003eB\u003c/strong\u003e) In-vitro screening for FORC growth inhibition with cell-free supernatants of compost-induced Streptomyces isolates CC6 and CC27. FORC treated with bacterial media (TSB) was used as the control. Growth was quantified by the change in GFP fluorescence (arbitrary units) monitored over 24h of incubation and normalized to the initial fluorescence measurements before estimation of the area under the growth curve (AUC). Statistical analysis was conducted using Dunnett’s multiple comparison test to compare the growth of FORC treated with Streptomyces CC6 and CC27 extracts against TSB media control. * denotes P \u0026lt; 0.05 and ns denotes P \u0026gt; 0.05 (6 technical replicates). \u0026nbsp;(\u003cstrong\u003eC\u003c/strong\u003e) Dual culture plate assay of isolates CC6 and CC27 with FORC.\u003cstrong\u003e \u003c/strong\u003eImages were taken 7 days after inoculation with FORC.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-5349913/v1/2365aaf0354adc236a79931e.png"},{"id":83067946,"identity":"fbb1ad59-bb5d-45ba-8751-2363bf401f31","added_by":"auto","created_at":"2025-05-19 16:08:31","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2999322,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5349913/v1/cb670334-bca8-4a9c-94d2-beb0966e3a89.pdf"},{"id":68562991,"identity":"0a41b803-86f2-46f2-bad4-a0d160411475","added_by":"auto","created_at":"2024-11-08 14:39:26","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1612223,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdditional file 1\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFigure S1. \u003c/strong\u003eAlpha diversity (observed OTUs) \u003cstrong\u003e(A)\u003c/strong\u003e,\u003cstrong\u003e \u003c/strong\u003eFaith’s phylogenetic diversity\u003cstrong\u003e (B)\u003c/strong\u003e and Pielou evenness index \u003cstrong\u003e(C) \u003c/strong\u003eof bacterial communities in the rhizosphere of compost amended and non-amended plants with and without FORC inoculation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFigure S2.\u003c/strong\u003e \u003cstrong\u003e(A)\u003c/strong\u003e Alpha diversity (observed OTUs) of fungal communities. \u003cstrong\u003e(B)\u003c/strong\u003e relative abundance of fungal ITS ASVs taxonomically classified to genus level, \u003cstrong\u003e(C)\u003c/strong\u003e relative abundance of ITS ASV taxonomically classified as Fusarium genus and most abundant in FORC-inoculated samples with and without compost, \u003cstrong\u003e(D)\u003c/strong\u003e percentage of shotgun metagenomics reads mapped to the genome of \u003cem\u003eF. oxysporum f. sp. radicis-cucumerinum\u003c/em\u003e (FORC).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFigure S3. \u003c/strong\u003ePhylogenetic tree and abundance of bins assembled from FORC-inoculated, compost amended and non-amended samples.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFigure S4. \u003c/strong\u003eAbundance of \u003cem\u003eActinomycetota\u003c/em\u003eand \u003cem\u003eBacillota\u003c/em\u003e associated MAGs assembled from FORC-inoculated, compost amended and non-amended samples and classified to genus level.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFigure S5. \u003c/strong\u003eAlpha diversity (observed and shannon) \u003cstrong\u003e(A)\u003c/strong\u003e of BGCs identified in the rhizosphere metagenomes of FORC-inoculated compost amended and non-amended plants. Phylum-level taxonomy of identified (by antiSMASH-V6.0) secondary metabolite-encoding biosynthetic gene clusters (BGCs) based on analysis of shotgun metagenome contigs (\u0026gt;5kbp) from compost-amended and non-amended FORC-inoculated cucumber rhizosphere samples \u003cstrong\u003e(B)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFigure S6. (A) \u003c/strong\u003eRelative abundance of 16S rRNA gene amplicon ASVs classified as Streptomyces. Relative abundance is based on 16S rRNA gene amplicon count table for all samples. (\u003cstrong\u003eB\u003c/strong\u003e) Progressive Mauve alignment of the Streptomyces CC7 isolate genome and the Streptomyces bin2.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFigure S7. \u003c/strong\u003eBiG-SCAPE network of BGCs identified in \u003cem\u003eStreptomyces\u003c/em\u003eisolates (CC6 and CC27) and bin2 genomes in comparison to the known BGCs in the MiBIG database.\u003c/p\u003e","description":"","filename":"AmutuhaireetalAdditionalfile1.docx","url":"https://assets-eu.researchsquare.com/files/rs-5349913/v1/a2c1f5e9ae54f1d04bb5cd75.docx"},{"id":68564064,"identity":"8e29624a-8a83-4582-b12e-4cb3c587e3d9","added_by":"auto","created_at":"2024-11-08 14:47:26","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":83807,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdditional file 2\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable S1. \u003c/strong\u003eSummary of 16S rRNA and ITS amplicon sequencing reads.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable S2. \u003c/strong\u003eSummary of shotgun metagenome generated reads and assembly.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable S3. \u003c/strong\u003eList of all\u003cstrong\u003e \u003c/strong\u003eAntiSMASH identified BGCs within pooled samples compost amended and non-amended samples.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable S4. \u003c/strong\u003eList of \u003cem\u003eActinomycetota\u003c/em\u003eand \u003cem\u003eBacillota\u003c/em\u003e NRPS and PKs BGCs annotated using MiBiG.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable S5. \u003c/strong\u003eSummary statistics of all generated MAGs binned from compost amended and non-amended samples.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable S6. \u003c/strong\u003eOrtho ANI values of \u003cem\u003eStreptomyces\u003c/em\u003e MAG bin2 and isolates CC6 and CC27.\u003c/p\u003e","description":"","filename":"AmutuhaireetalAdditionalfile2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5349913/v1/e7e2dc39bd4751bbbb00ba59.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Identifying root-associated bacteria and potential mechanisms linked to compost suppressiveness towards Fusarium oxysporum.","fulltext":[{"header":"Background","content":"\u003cp\u003eSoilborne fungal phytopathogens pose a significant threat to global food security because they infect a wide range of crops and are difficult to control [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e] especially since most of them can survive in soil in the absence of host plants [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. The \u003cem\u003eFusarium oxysporum\u003c/em\u003e species complex includes several strains that cause wilt or root and crown rot in a variety of commercially significant crops [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. These pathogens are particularly challenging for plant protection management [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e] due to growing resistance of some Fusarium species to commonly used fungicides, such as azoles [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Chemical fungicides are often the most effective way to control soil borne fungal pathogens [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], but increased regulation due to human and environmental health ramifications has strongly restricted use of several conventionally applied compounds [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. This phenomenon, coupled to increasing global demand for food production and the predicted geographic expansion of phytopathogens due to climate change, has created a dire need for sustainable alternatives [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSoil amendments, including animal and green manure, organic wastes [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], compost, [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] and more recently, biochar [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] are considered to be beneficial strategies for controlling soilborne pathogens. The capacity of compost to effectively suppress soilborne fungal pathogens has been previously demonstrated in a diverse array of studies [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] and while abiotic factors such as the presence of compost-derived fungitoxic chemicals have been reported, collective evaluation of current research suggests that the primary mechanisms behind compost disease suppressiveness are linked to shifts in the overall soil microbial community composition, diversity, activity and functioning [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], that facilitate enrichment of specific groups of bacteria with antagonistic activity against soilborne pathogens [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe rhizosphere microbiome enhances plant nutrient uptake, facilitates protection against abiotic stress, and acts as the first line of defense against soilborne fungal pathogens [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Certain rhizosphere bacteria protect plant roots from colonization by soilborne pathogens through a range of mechanisms, including competition for space and nutrients, parasitism, induction of systemic resistance [\u003cspan additionalcitationids=\"CR23\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. In addition, various root-associated bacteria produce an array of small molecules known as secondary metabolites (SMs) that play vital ecological roles in interactions with the local microbiome, plant roots and soilborne pathogens, and subsequently impact plant growth and health [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Non-ribosomal peptides (NRPs) and polyketides (PKs) are among the most prevalent classes of SMs across the three life domains [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. These compounds include iron scavenging siderophores, stress protectant pigments, signaling molecules, and antimicrobials, including antifungals [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. NRPs and PKs are synthesized by non-ribosomal peptide synthetases (NRPS) and polyketide synthases (PKS) respectively [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], huge enzymatic complexes (typically more than 500 KDa) that are encoded by large biosynthetic gene clusters (BGCs). These complexes have a similar multi-modular architecture, consisting of repeating domains that are each in charge of incorporating a single building block into an expanding molecule in a stepwise fashion. The ability of NRPS and PKS to incorporate a wide range of building blocks enables them to assemble and customize a diverse array of chemical compounds with a variety of properties and functions [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], making them an untapped source of potentially novel antifungal compounds for application in agriculture as biofungicides.\u003c/p\u003e \u003cp\u003eWhile numerous studies have demonstrated compost-facilitated disease suppressiveness in various pathosystems and identified bacterial taxa that are stimulated by compost amendment [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], few have deliberated the mechanisms responsible for disease suppression by compost-induced bacteria [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. We hypothesize that compost enhances disease suppression by stimulating rhizosphere bacteria that antagonize pathogens at least partially through the production of antimicrobial secondary metabolites.\u003c/p\u003e \u003cp\u003eThis study aimed to identify microbial populations and associated secondary metabolite encoding genes involved in compost-induced soilborne pathogen suppression, using a model comprised of greenhouse grown cucumber plants inoculated with \u003cem\u003eF. oxysporum f. sp. radicis-cucumerinum\u003c/em\u003e (FORC). Initially, we evaluated the impact of compost amendment on the microbial community composition of cucumber rhizospheres (with and without FORC inoculation) using 16S rRNA gene amplicon sequencing and identified specific bacterial populations that were potentially associated with the observed compost-induced suppressiveness. Subsequently, we identified functional genes potentially linked to compost facilitated suppressiveness using shotgun metagenomics, specifically focusing on genes encoding for secondary metabolites from compost stimulated bacterial taxa. Using culturomics, we isolated representative bacteria associated with compost-suppressiveness and tested their capacity to secrete FORC-antagonizing extracellular metabolites \u003cem\u003ein-vitro.\u003c/em\u003e\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eGreenhouse experiment\u003c/h2\u003e \u003cp\u003eFor compost amended treatments, soil, commercial perlite (Perlite 212 Agrekal, Israel), and compost (Azur nurseries, Israel) were mixed in a 2:2:1 (v/v) ratio, whereas in non-amended treatments, soil was mixed with perlite in a 1:1 (v/v) ration. These soil mixtures were watered every other day for a week before transplanting seedlings (see below). A clay-rich soil from Ahituv, Israel (32.389988, 34.990965) used exclusively for growing cucumbers in greenhouses was used in this study. It was air-dried at room temperature, and then ground and sieved through a 2 mm mesh to ensure uniform particle size. Cucumber (\u003cem\u003eCucumis sativus\u003c/em\u003e) seeds (Manny- Genesis seeds, lot # 10614C) were sown in seedling trays filled with perlite:vermiculite mixture (1:3, respectively) and the trays were incubated in a temperature-controlled growth chamber (28\u0026ndash;30\u0026deg;C) for 7 days and were watered every other day with tap water to facilitate germination. Germinated seedlings were then transplanted into 2.5-inch square black nursery pots (one plant per pot) containing the potting mixtures described above, producing the four following treatments: (i) non-inoculated, non-amended (5 plants), (ii) non-inoculated, compost-amended (5 plants), (iii) FORC-inoculated, non-amended (6 plants), and (iv) FORC-inoculated, compost-amended (6 plants). The inoculated treatments contained 1% FORC (w/w), prepared according to the protocol previously described by [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. All plants were fertigated with 500X diluted commercial greenhouse liquid fertilizer for 21 days.\u003c/p\u003e \u003cp\u003eRhizosphere samples were collected by gently shaking off excess soil from the roots and collecting the remaining soil that was firmly adhered to the root surface. Half of each soil was stored in sterile 50 ml polypropylene tubes at -80\u003csup\u003eo\u003c/sup\u003eC for DNA extraction, whereas the other half was mixed in glycerol (final concentration of 50%) and stored at -80\u003csup\u003eo\u003c/sup\u003e C for subsequent strain isolation described below.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eDisease phenotyping\u003c/h3\u003e\n\u003cp\u003eTo determine the capacity of compost to induce disease suppression, disease incidence, stem height, shoot dry weight, and root fresh weight were measured at 21 days post-inoculation. Disease incidence was determined by counting the number of wilted plants, shoot dry weight (g) was measured following overnight drying (100\u003csup\u003eo\u003c/sup\u003eC) of plant excised shoots, and root fresh weight by measuring the weight of roots (g) after collection of rhizosphere soil samples.\u003c/p\u003e\n\u003ch3\u003eRhizosphere sampling, DNA extraction, and sequencing\u003c/h3\u003e\n\u003cp\u003e DNA was extracted from 0.25 g of rhizosphere material of all plants (N\u0026thinsp;=\u0026thinsp;22) using the DNeasy PowerSoil Kit (Qiagen, USA), according to the manufacturer's protocol. For characterization of bacterial and fungal communities, we PCR amplified the V3-V4 region of the 16S rRNA gene (bacteria) and the nuclear ribosomal internal transcribed spacer (ITS, fungi), using the 341F/806R [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e] and ITS1-F/ITS2R [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e] primer sets, respectively. The primers were appended with CS1(forward), and CS2 (reverse) Illumina sequencing adapters and sequencing was conducted using a paired-end 2x300bp cycle run on an Illumina MiSeq sequencing system using MiseqV3 chemistry, at Rush University Genomics and Microbiome Core Facility (Chicago, USA).\u003c/p\u003e \u003cp\u003eShotgun metagenomic sequencing was conducted on 12 DNA samples from the FORC-inoculated treatments (6 non-amended and 6 compost-amended) using an Illumina NovaSeq S4 platform with 2x150bp paired-end reads, employing Illumina dsDNA shotgun library preparation. The sequencing was performed at Rush University Genomics Microbiome Core Facility.\u003c/p\u003e \u003cp\u003eAll datasets have been deposited in the NCBI Short Read Archive under \u003cb\u003eBioproject PRJNA1169419\u003c/b\u003e.\u003c/p\u003e\n\u003ch3\u003eBioinformatic analysis of generated amplicon sequences\u003c/h3\u003e\n\u003cp\u003eBacterial 16S rRNA gene and fungal ITS amplicons were demultiplexed and analyzed using QIIME2 (v2019.07) [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e], after applying the DADA2 denoising pipeline [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e] (\u003cb\u003eTable \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e, Additional file 2\u003c/b\u003e). Amplicon Sequence Variants (ASVs) with a frequency of less than 3 were removed. Taxonomic assignment of the ASVs was done using the QIIME2 q2-feature-classifier with the Silva 138 [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e] and UNITE [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e] databases, for bacterial and fungal ASV characterizations, respectively. A mid-point rooted phylogenetic tree for the calculation of diversity matrices was created with fasttree and mafft alignment (QIIME2) using the generated ASVs. Raw count tables, ASV taxonomy, and the rooted phylogenetic tree generated in QIIME2 were exported for downstream analyses and visualization in R (version 4.3.1).\u003c/p\u003e\n\u003ch3\u003eQuality control and assembly of shotgun metagenomic reads and characterization of assembled contigs\u003c/h3\u003e\n\u003cp\u003eThe quality of raw shotgun metagenomic reads was assessed using FASTQC, and low-quality reads and adapters were removed using Trimmomatic [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e] with default parameters for adapter trimming, and CROP:145 HEADCROP:15 SLIDINGWINDOW:4:20 MINLEN:100 parameters for low quality trimming. Subsequently, the reads were aligned against the FORC reference genome (GenBank GCA_001702695.2) using Bowtie2, --very-sensitive-local flag [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e] and FORC reads were removed using SAMtools [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. High quality FORC-filtered reads from all samples were pooled and assembled using MEGAHIT [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e] (\u003cb\u003eTable \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e, Additional file 2\u003c/b\u003e). The assembled contigs were filtered to retain contigs\u0026thinsp;\u0026gt;\u0026thinsp;2000bp, and these were used as input for prediction of open reading frames (ORFs) using Prodigal [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e], in sensitive mode. The predicted ORFs were aligned to the NCBI-nr protein database using DIAMOND [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e], and MEGAN V6 Ultimate Edition [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e] was then used for taxonomic assignment of these ORFs based on the Lowest common ancestor (LCA).\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eDetection and annotation of biosynthetic gene clusters (BGCs) from assembled shotgun metagenomic contigs\u003c/h2\u003e \u003cp\u003eAssembled contigs from compost-amended and non-amended FORC-inoculated treatments were filtered to maintain contigs that were \u0026gt;\u0026thinsp;5000bp length, which were used to identify predicted secondary metabolite encoding BGCs with antiSMASH v6 [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e], using the following parameters: \u0026ndash;cb-general \u0026ndash;cb-subclusters \u0026ndash;pfam2go \u0026ndash;strictness relaxed \u0026ndash;genefinding-tool prodigal-m mode. A biom table of reads mapping to BGC core regions was generated using BiG-MAP [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e], and differential abundance analysis of compost amended and nonamended groups was performed using Maaslin2 [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e] using Total Sum Scaling (TSS) normalized table. The taxonomy of the identified BGCs was assigned by aligning contigs to the NCBI nonredundant database using DIAMOND and LCA classification with MEGAN [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. NRPS and PKS BGCs were aligned against the MiBiG 3.0 [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e] database to predict their putative functions using DIAMOND [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e] (cutoffs: e-value\u0026thinsp;\u0026lt;\u0026thinsp;10\u003csup\u003e\u0026minus;\u0026thinsp;40\u003c/sup\u003e and identity\u0026thinsp;\u0026gt;\u0026thinsp;50%), as previously described [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMAG binning and annotation\u003c/h3\u003e\n\u003cp\u003eContigs were binned using metaWRAP v1.3.2 [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e] with a minimum completeness of 75% and a maximum contamination of 5%. Binning was performed using MaxBin2 [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e], metaBAT2 [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e], and CONCOCT [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. The resulting bins were dereplicated using dRep [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e] v3.4.2 with default parameters. Taxonomic classification was achieved using the GTDB-Toolkit [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e] v2.3.2 (GTDB-Tk) classify workflow.\u003c/p\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eStatistical differences in shoot dry weight and root fresh weight between treatments were evaluated using one-way ANOVA and multiple comparisons of means using the Tukey-Kramer (HSD) test (α\u0026thinsp;=\u0026thinsp;0.05) in JMP software (JMP\u0026reg;, Version 17. SAS Institute Inc., Cary, NC, 1989\u0026ndash;2023). Shannon, OTU richness and Bray-Curtis diversity indices (for evaluating alpha- and beta-diversity of bacterial and fungal communities, respectively) were calculated using the Vegan R package. Differential abundance analysis of amplicon-generated count tables was performed using MaASlin2 [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e] and Lefse [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eBacteria isolation, identification and whole-genome sequencing\u003c/h2\u003e \u003cp\u003eSelected bacteria were isolated from glycerol stocks of compost-induced disease suppressed rhizosphere samples using ccucumber plant-extract media prepared according to [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e] with several modifications. Briefly, 20g of cucumber shoots were washed in tap water to remove any soil particles, cut into small pieces, and blended with minimum amount of water in a waring blender. The resulting extract was filtered through a triple layered gauze and stored at 4\u003csup\u003eo\u003c/sup\u003eC as a stock solution. Agar plates were prepared by diluting the cucumber extract with distilled water in a ratio of 1:10 (v/v) and addition of 1.5% (w/v) Bacto agar (Difco Laboratories, Detroit). The medium was sterilized by autoclaving at 121\u003csup\u003eo\u003c/sup\u003eC for 30 minutes and 50 mg/l of cycloheximide was added to preclude the growth of fungi. Cultures were incubated for 14 days at 30\u003csup\u003eo\u003c/sup\u003eC, and the isolated bacteria were taxonomically identified based on 16S rRNA gene amplicon sequencing using primers; 11F (GTTTGATCCTGGCTCAG) and 1392R (ACGGGCGGTGTGTRC) [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. The PCR products were sequenced by Macrogen Inc. (Seoul, Korea) and aligned against the NCBI nr database using BLASTn.\u003c/p\u003e \u003cp\u003eHigh molecular weight genomic DNA was isolated from \u003cem\u003eStreptomyces\u003c/em\u003e CC6 and CC27 isolates using MagAttract HMW DNA (Qiagen, USA) extraction Kit following the manufacturer\u0026rsquo;s protocol. Hybrid whole genome sequencing and assembly were performed by Plasmidsaurus Inc. (USA) service using Oxford Nanopore and Illumina Sequencing Technologies.\u003c/p\u003e \u003cp\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eScreening for\u003c/span\u003e \u003cspan type=\"ItalicUnderline\" class=\"ItalicUnderline\" name=\"Emphasis\"\u003ein-vitro\u003c/span\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eantifungal activity.\u003c/span\u003e\u003c/p\u003e \u003cp\u003eGreen fluorescent protein-labeled (gfp) FORC [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e] was exposed to cell-free supernatants (CFS) from \u003cem\u003eStreptomyces\u003c/em\u003e CC6 and CC27 to assess potential presence of inhibitory secreted metabolites, using the methodology developed by [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]. Briefly, CFS were prepared from bacterial spent media as follows; one milliliter of 3 days old starter culture was used to inoculate 50 ml of Tryptic Soy Broth (TSB, BD Bacto\u0026trade; Soybean-Casein Digest Medium) in 250ml Erlenmeyer flasks. The flasks were incubated for 7 days at 30\u003csup\u003eo\u003c/sup\u003eC and 170 rpm. The bacterial spent media was then centrifuged (5000g for 10 minutes) and the supernatant was filtered using a 0.22\u0026micro;m Millex-HV syringe filter (Sigma-Aldrich, Israel).\u003c/p\u003e \u003cp\u003eSpore solution was prepared by inoculating a plug of gfp-labeled FORC mycelia taken from a 5 days old PDA (potato dextrose agar) plate in 25ml of fresh PDB (potato dextrose broth) media in 250ml Erlenmeyer flasks with and flasks were incubated for 7 days 25\u003csup\u003eo\u003c/sup\u003eC and shaking at 100 rpm. FORC culture was filtered through 40 \u0026micro;m filters, and concentration was determined by counting spores using a Hemocytometer. \u003cem\u003eIn-vitro\u003c/em\u003e antifungal assays were carried out in 96-microwell plates by adding 100\u0026micro;l of bacterial cell-free supernatant (CFS) into 100\u0026micro;l of gfp-labelled FORC spores (10\u003csup\u003e5\u003c/sup\u003e spores/ml). CFS-treated FORC spores were incubated at 25\u0026deg;C for 24 h, and growth changes were monitored by measuring fluorescence and OD using the multimode Microplate Reader (Synergy H1, BioTek, USA).\u003c/p\u003e \u003cp\u003eFor dual culture plate assay, \u003cem\u003eStreptomyces\u003c/em\u003e CC6 and CC27 starter cultures were grown on LB medium (Luria-Bertani) for 3 days. Ten microliters of the bacteria culture were spot inoculated in quadruplets on half strength LB agar plates, 2cm from the center of the plate. Plates were incubated at 30\u003csup\u003eo\u003c/sup\u003eC for 5 days and thereafter a plug of 7 days old FORC mycelium was placed in the center of plate. The plates co-inoculated with bacterial and fungi were incubated at 28\u003csup\u003eo\u003c/sup\u003eC and visually monitored for a period of 7 days.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eCompost amendment alleviates FORC-induced disease in cucumbers\u003c/h2\u003e \u003cp\u003eDisease incidence in FORC-inoculated cucumber plants was determined by counting the number of wilted plants in soil with and without compost at 21 days post inoculation. No mortality was recorded for inoculated plants grown in compost-amended soil, while 50% mortality (N\u0026thinsp;=\u0026thinsp;6) was observed in inoculated plants grown in non-amended soil (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Additionally, FORC inoculation significantly reduced shoot dry weight and root fresh weight of inoculated cucumber plants grown in compost-free soil (Tukey HSD test, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), whereas these values were not higher in inoculated plants grown in compost amended soil relative to control (pathogen free) plants with and without compost amendment (Tukey HSD test, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u003cb\u003e).\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eCompost amendment reduces the relative abundance of Fusarium, and alleviates FORC-induced reduction in rhizosphere microbial diversity and shifts in community composition\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWe evaluated the impact of compost amendment on the rhizosphere bacterial and fungal diversity and community composition in FORC-inoculated and non-inoculated plants using fungal ITS and bacterial 16S rRNA gene amplicon sequencing.\u003c/p\u003e \u003cp\u003eWe did not observe significant differences in bacterial community richness and phylogenetic diversity (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05, Tukey HSD; \u003cb\u003eFigure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA and S1B, Additional file 1\u003c/b\u003e) across all groups (with and without compost, in both the presence and absence of FORC). However, FORC inoculation significantly decreased bacterial species evenness (by 2.7%; Pielou's evenness index) compared to non-inoculated plants without compost (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Tukey HSD). Pathogen inoculation caused a 1.56% reduction in the bacterial species evenness in compost amended plants when compared to non-inoculated plants without compost, however this was not statistically significant, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05, Tukey HSD (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, \u003cb\u003eand Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eC, Pielou's evenness index, Additional file 1\u003c/b\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWithout FORC, compost amendment slightly reduced fungal diversity in the rhizosphere. However, without compost FORC inoculation facilitated a 32% (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Tukey HSD) decrease in fungal diversity, whereas in compost amended samples the decrease was only 13% and was not significant compared to the compost-amended samples not inoculated with FORC (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, and \u003cb\u003eFigure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eA, Additional file 1\u003c/b\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eDistinct bacterial and fungal communities were observed in compost amended \u003cem\u003evs.\u003c/em\u003e non-amended cucumber plants (Bray-Curtis Dissimilarity matrix), and the amendment group explained 59% and 53.6% (Adonis test - amendment group R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.59 and 0.536, bacterial 16S rRNA and fungal ITS respectively, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) of the differences in bacterial and fungal communities respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eC and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). In the absence of compost, bacterial and fungal communities were significantly affected by FORC inoculation, as opposed to the compost amended communities in which FORC-induced shifts were considerably less pronounced. Estimation of the relative abundance of FORC-associated ITS amplicon sequence variants (ASV) and of FORC-characterized reads from shotgun metagenomic analyses, indicated that compost amendment significantly reduced the relative abundance of FORC in the rhizosphere (\u003cb\u003eFigure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eB, S2C and S2D, Additional file 1\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eIn summary, compost significantly reduced fungal diversity but did not have notable impact on bacterial diversity. It significantly altered the composition of both bacterial and fungal communities in addition to mitigating the negative impact of FORC inoculation by reducing FORC abundance and minimizing microbial community shifts in the rhizospheres of FORC inoculated cucumber plants. Considering the trends described above, previous reports suggesting bacteria are involved in suppressiveness and the vast reservoir of unexplored antifungal compounds produced by rhizosphere bacteria, we chose to hereafter focus on the bacterial community.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eCompost amendment mitigates FORC-induced reduction in the relative abundance of the\u003c/b\u003e \u003cb\u003eActinomycetota\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003eBacillota\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo identify bacterial populations potentially involved in facilitating compost suppressiveness, we taxonomically annotated the rhizosphere communities in all treatments and performed differential abundance analysis on the 16S rRNA gene amplicon sequencing data using LEfSe.\u003c/p\u003e \u003cp\u003eFORC inoculation significantly reduced the relative abundance of \u003cem\u003eBacillota\u003c/em\u003e and \u003cem\u003eActinomycetota\u003c/em\u003e in the absence of compost amendment, but this reduction was not significant in the compost-amended treatments. Specifically, in FORC inoculated cucumber plants the relative abundance of the \u003cem\u003eBacillota Thermoactinomycetaceae\u003c/em\u003e, \u003cem\u003eClostridiales\u003c/em\u003e Family_XI, and \u003cem\u003ePeptococcaceae\u003c/em\u003e, as well as \u003cem\u003eActinomycetota\u003c/em\u003e families \u003cem\u003eStreptomycetaceae\u003c/em\u003e, \u003cem\u003eThermomonosporaceae\u003c/em\u003e, \u003cem\u003eGlycomycetaceae\u003c/em\u003e, \u003cem\u003ePropionibacteriaceae\u003c/em\u003e, \u003cem\u003eNocardiopsaceae\u003c/em\u003e, \u003cem\u003eIntrasporangiaceae\u003c/em\u003e, \u003cem\u003ePseudonorcadiaceae\u003c/em\u003e and unclassified \u003cem\u003eMicrococcales\u003c/em\u003e families was significantly higher in the rhizosphere of the compost-amended treatments than in the plants that were not amended with compost \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eTo validate 16S rRNA gene amplicon results and achieve high-resolution taxonomic classification, we performed shotgun metagenomics on FORC-inoculated samples (with and without compost), binning 116 medium- to high-quality dereplicated MAGs (\u0026gt;\u0026thinsp;70% completion, \u0026lt; 5% contamination) using the metaWRAP pipeline. Taxonomic classification (using the GTDB_tk classify workflow) revealed that MAGs from \u003cem\u003eActinomycetota\u003c/em\u003e and \u003cem\u003eBacillota\u003c/em\u003e were significantly associated with compost amendment (\u003cb\u003eTable S5, Additional file 2 and Figure S3, Additional file 1\u003c/b\u003e). These included genera such as \u003cem\u003eStreptomyces\u003c/em\u003e, \u003cem\u003eActinomadura\u003c/em\u003e, \u003cem\u003eSaccharomonospora\u003c/em\u003e, \u003cem\u003ePseudonocardia\u003c/em\u003e, \u003cem\u003eGlycomyces\u003c/em\u003e, \u003cem\u003eThermobifida\u003c/em\u003e, \u003cem\u003ePlanifilum\u003c/em\u003e, and \u003cem\u003eNovibacillus\u003c/em\u003e, (\u003cb\u003eFigure S4, Additional file 1\u003c/b\u003e), consistent with the compost-associated families identified in the 16S rRNA gene amplicon analysis. Collectively, these results suggest that compost amendment mitigated FORC-induced dysbiosis of the rhizosphere microbiome, maintaining high relative abundance of specific \u003cem\u003eBacillota\u003c/em\u003e and \u003cem\u003eActinomycetota\u003c/em\u003e genera.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eActinomycetota\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003eBacillota\u003c/b\u003e \u003cb\u003eassociated SM-encoding gene clusters are highly abundant in disease suppressed compost-amended cucumber rhizospheres.\u003c/b\u003e\u003c/p\u003e \u003cp\u003eWe postulated that the compost-facilitated suppressiveness and alleviation of FORC-induced dysbiosis, was at least partially linked to bacterial secondary metabolites that antagonize FORC. To identify secondary metabolite-encoding gene clusters, contigs larger than 5000bp were pooled and used as input for antiSMASH v6 and BiG-MAP tool was used to generate gene cluster abundance table, focusing only on the core genes of a BGC.\u003c/p\u003e \u003cp\u003eIn total, 1237 BGCs were identified in FORC inoculated samples with and without compost (\u003cb\u003eTable S3, Additional file 2\u003c/b\u003e). While no differences in the BGCs diversity (Shannon index: \u003cb\u003eFigure S5A, Additional file 1\u003c/b\u003e) were observed between compost amended and non-amended samples, there was a significant difference in the BGC composition between these two groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). BGCs phylogenetically linked to \u003cem\u003eActinomycetota\u003c/em\u003e and \u003cem\u003eBacillota\u003c/em\u003e were significantly more abundant in disease suppressed compost amended cucumber rhizospheres, while \u003cem\u003ePseudomonadota\u003c/em\u003e and \u003cem\u003eBacteroidota\u003c/em\u003e BGCs were more prevalent in non-amended FORC-inoculated rhizospheres \u003cb\u003e(Figure S5B, Additional file 1\u003c/b\u003e). Compost-enriched BGCs were mainly taxonomically associated with the \u003cem\u003eActinomycetota\u003c/em\u003e families \u003cem\u003eStreptomycetaceae, Thermomonosporaceae, Pseudonorcadiaceae, Glycomycetaceae\u003c/em\u003e, and \u003cem\u003eNocardiopsaceae\u003c/em\u003e, and the \u003cem\u003eBacillota\u003c/em\u003e family \u003cem\u003eThermoactinomycetaceae\u003c/em\u003e, corresponding to the results of 16S rRNA gene amplicon analysis \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003eB\u003cb\u003e).\u003c/b\u003e The compost \u0026ldquo;protected\u0026rdquo; bacterial families harbor diverse classes of SM-encoding BGCs including NRPS, PKS (T1, T2 and T3), siderophores, betalacones, lanthipeptides (classes i-iii), ectoine and terpenes, with \u003cem\u003eStreptomycetaceae\u003c/em\u003e harboring the most abundant and diverse collection of SMs \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003eC\u003cb\u003e)\u003c/b\u003e. In summary, although the diversity of BGCs was similar in compost-amended and non-amended FORC-inoculated rhizospheres, their composition differed significantly. The rhizospheres of disease-suppressed, compost-amended plants were enriched in a variety of BGCs from the \u003cem\u003eActinomycetota\u003c/em\u003e and \u003cem\u003eBacillota\u003c/em\u003e families, including \u003cem\u003eStreptomycetaceae\u003c/em\u003e, \u003cem\u003eThermomonosporaceae\u003c/em\u003e, \u003cem\u003ePseudonocardiaceae\u003c/em\u003e, \u003cem\u003eGlycomycetaceae\u003c/em\u003e, \u003cem\u003eNocardiopsaceae\u003c/em\u003e, and \u003cem\u003eThermoactinomycetaceae\u003c/em\u003e, consistent with the results of 16S rRNA gene amplicon analysis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eCompost stabilized bacteria possess NRPS and PKS BGCs encoding for antimicrobial compounds and siderophores\u003c/h2\u003e \u003cp\u003eNPRS and PKS secondary metabolites are known to play important ecological roles in microbe-microbe and microbe-plant interactions in soil and roots microbiomes. To shed light on the potential functions of the identified NRPS and PKS gene clusters in compost amended, FORC suppressive rhizospheres, we focused on NRPS and PKS BGCs from the \u003cem\u003eActinomycetota\u003c/em\u003e and \u003cem\u003eBacillota\u003c/em\u003e taxa, due to their profuseness in these samples. A total of 160 NRPS- and PKS-encoding BGCs were aligned against the MIBiG database using Diamond blastx and function was assigned if a BGC had an E-value\u0026thinsp;\u0026lt;\u0026thinsp;10\u003csup\u003e\u0026minus;\u0026thinsp;40\u003c/sup\u003e and \u0026gt;\u0026thinsp;50% similarity to a documented secondary metabolite producing cluster [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. In total, 118 out of the 160 identified NRPS/PKS BGCs displayed statistically significant differences (MaAsLin2, \u003cem\u003eP\u003c/em\u003e adj\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in abundance between the compost-amended and non-amended treatments (\u003cb\u003eTable S4, Additional file 2)\u003c/b\u003e, 47% (56/118) of whom were functionally categorized. The functionally annotated NRPS and PKS BGCs were primarily phylogenetically associated with the \u003cem\u003eActinomycetota\u003c/em\u003e phylum, in contrast to those affiliated with \u003cem\u003eBacillota\u003c/em\u003e phylum (particularly; \u003cem\u003eThermoactinomycetaceae\u003c/em\u003e, unclassified \u003cem\u003eNegativicutes\u003c/em\u003e and \u003cem\u003eBacillales\u003c/em\u003e) that could not be assigned a functional role according to our cutoff criteria. The annotated NRPS and PKS BGCs were primarily linked to production of antimicrobial compounds and siderophores (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e5\u003c/span\u003eA and \u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). The antimicrobial encoding NRPS and PKS BGCs in disease suppressed, compost amended samples were predominantly associated with the \u003cem\u003eStreptomyces\u003c/em\u003e genus. These included NRPS BGCs encoding for atratumycin, enduracidin, cadaside A, and maduralactomycin A antimicrobials; and PKS BGCs encoding for concanamycin A, azalomycin F3a, A-47934, and diazepinomicin antimicrobials (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e5\u003c/span\u003eA and \u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Other notable antimicrobial encoding BGCs include: \u003cem\u003eActinomadura\u003c/em\u003e-associated NRPS and PKS BGCs encoding for coprisamide C and merochlorin A, respectively; the NRPS taromycin A gene cluster from \u003cem\u003eSaccharomonospora\u003c/em\u003e; PKS loseolamycin A1; and griseorhodin A from \u003cem\u003eGlycomyces\u003c/em\u003e and \u003cem\u003eThermobifida\u003c/em\u003e. Siderophores were mainly encoded by NRPS BGCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Additionally, NRPS gene clusters encoding for siderophore production were found in compost-stimulated \u003cem\u003eSaccharomonospora\u003c/em\u003e (mirubactin), \u003cem\u003eThermobifida\u003c/em\u003e (fuscachelin A), \u003cem\u003ePseudonocardiaceae\u003c/em\u003e (saccharochelin A and albachelin), and \u003cem\u003eActinomadura\u003c/em\u003e (saccharochelin A) families, but not in \u003cem\u003eStreptomyces\u003c/em\u003e. Overall, NRPS and PKS BGCs enriched in compost-amended, FORC-inoculated rhizospheres were primarily associated with antimicrobial and siderophore production. Most antimicrobial BGCs were associated with the \u003cem\u003eStreptomyces\u003c/em\u003e genus, and the majority of siderophore encoding BGCs belonged to the \u003cem\u003eSaccharomonospora\u003c/em\u003e, \u003cem\u003eThermobifida\u003c/em\u003e, and \u003cem\u003eActinomadura\u003c/em\u003e genera.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eCompost stimulated\u003c/b\u003e \u003cb\u003eStreptomyces\u003c/b\u003e \u003cb\u003eexcrete FORC-antagonizing metabolites\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe contig-based functional annotation of NRPS and PKS BGCs highlighted the \u003cem\u003eStreptomycetaceae\u003c/em\u003e family as a potential producer of antimicrobial compounds in the compost-amended FORC inoculated samples. Using plant-based medium, we isolated two \u003cem\u003eStreptomyces\u003c/em\u003e strains (CC6 and CC27) from compost induced, disease suppressed rhizosphere samples. 16S rRNA gene analysis confirmed that these strains clustered with the \u003cem\u003eStreptomyces\u003c/em\u003e ASV_2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e6\u003c/span\u003eA), whose abundance was slightly augmented by compost in the presence of FORC, in contrast to plants not amended with compost where it was significantly lower in FORC inoculated samples (\u003cb\u003eFigure S6A, Additional file 1\u003c/b\u003e). Additionally, the genomes of these two isolates were over 99% identical to the \u003cem\u003eStreptomyces\u003c/em\u003e bin2 MAG (ortho ANI) (\u003cb\u003eTable S6, Additional file 2 and Figure S6B, Additional file 1\u003c/b\u003e), confirming isolation of these key strains.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe genomes of CC6, CC27, and bin2 share 11 unique BGCs not found in MIBiG, along with a transAT-PKS-NRPS cluster sharing 93% of its genes with the weishanmycin BGC and a HR-T2PKS-NRPS hybrid cluster sharing 55% of its genes with the ishigamide BGC (\u003cb\u003eFigure S7, Additional file 1\u003c/b\u003e). \u003cem\u003eStreptomyces\u003c/em\u003e CC6 and CC27 extracts significantly inhibited growth (\u003cem\u003ein-\u003c/em\u003evitro) of FORC (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). The fungal inhibitory phenotype was validated using a bacteria-fungal dual culture plate assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e6\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe ability of compost to suppress soilborne pathogens in various pathosystems is well established [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Evidence indicates that while physicochemical properties of compost contribute to its suppressiveness, sterilization leads to a loss of this effect, underscoring the crucial role of microorganisms [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e]. While various processes such as altering soil physicochemical characteristics to promote microbial growth and activity, and augmenting the native rhizosphere microbiome by introducing new microbes [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e, \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e] have been associated with compost-induced suppressiveness, the specific microbial populations and mechanisms driving compost induced disease suppression remain unclear. This study aimed to identify key rhizosphere bacterial communities and genes related to secondary metabolite production, potentially involved in compost-mediated suppression of FORC in greenhouse grown cucumbers.\u003c/p\u003e \u003cp\u003eGreenhouse experiments validated that compost amendment mitigated FORC-induced disease, and microbial analyses revealed that this phenomenon was linked to a significant reduction in the relative abundance of FORC in the rhizosphere. While compost amendment altered the rhizosphere microbial community composition when compared to non-amended samples, our findings indicate that it also \"stabilized\" the rhizosphere microbiome in the presence of FORC, mitigating pathogen-induced reduction of microbial diversity and alleviating shifts in community composition. Compost may stabilize the rhizosphere microbiome in the presence of a pathogen by altering the soil physicochemical characteristics such as pH and organic carbon to promote proliferation of the native microbiome or augmentation of the native microbial communities by introducing in new species [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWe propose that compost may facilitate disease suppression by alleviating pathogen-induced dysbiosis of the rhizosphere microbiome, maintaining robustness of specific bacterial populations. Our findings are supported by previous studies that found that compost amendment led to sustained alterations in the structure of soil microbial communities [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e, \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e], however these were all conducted without addition of pathogens. The compost stimulated eubiosis of the FORC-amended rhizosphere microbiome was most prominent in the case of selected \u003cem\u003eBacillota\u003c/em\u003e and \u003cem\u003eActinomycetota\u003c/em\u003e genera that were highly abundant in FORC inoculated plants with compost, but almost completely absent in the compost-free inoculated treatments. This is supported by past studies that found positive correlations between \u003cem\u003eActinomycetota\u003c/em\u003e and \u003cem\u003eBacillota\u003c/em\u003e abundance, and inhibition of soilborne pathogens such as \u003cem\u003eFusarium oxysporum\u003c/em\u003e [\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e, \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e] and \u003cem\u003eRalstonia solanacearum\u003c/em\u003e [\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e]. Interestingly, reduction of these phyla in tomato rhizosphere was associated with increased disease incidence [\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDelving into the 16S rRNA and MAG binning of shotgun metagenomic data revealed significantly higher relative abundance of \u003cem\u003eStreptomyces\u003c/em\u003e, \u003cem\u003eActinomadura\u003c/em\u003e, \u003cem\u003eSaccharomonospora\u003c/em\u003e, \u003cem\u003ePseudonocardia\u003c/em\u003e, \u003cem\u003eGlycomyces\u003c/em\u003e and \u003cem\u003eThermobifida\u003c/em\u003e (\u003cem\u003eActinomycetota\u003c/em\u003e) and of \u003cem\u003ePlanifilum\u003c/em\u003e and \u003cem\u003eNovibacillus\u003c/em\u003e (\u003cem\u003eBacillota\u003c/em\u003e) in FORC-inoculated rhizosphere samples amended with compost, compared to inoculated plants without compost. These genera were enriched with genes encoding production of diverse secondary metabolites including NRPS and PKS genes associated with production of antimicrobial and siderophore compounds, suggesting that they may directly antagonize soilborne pathogens through excreted antimicrobial and iron scavenging compounds. Previous studies indicated that \u003cem\u003eActinomycetota\u003c/em\u003e and \u003cem\u003eBacillota\u003c/em\u003e strains can directly antagonize Fusarium and other pathogens through production of antibiotics and lytic enzymes, and indirectly by competition such as production of siderophores [\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e, \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSecondary metabolites produced by non-ribosomal peptide synthetases (NRPS) and polyketide synthases (PKS) are known to play crucial roles in antagonizing fungal pathogens in the rhizosphere [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. \u003cem\u003eStreptomyces\u003c/em\u003e are known to produce a wide range of antimicrobial compounds and includes strains that have been shown to antagonize Fusarium wilts in several \u003cem\u003ein-planta\u003c/em\u003e studies [\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e, \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e]. We found that \u003cem\u003eStreptomyces\u003c/em\u003e spp. were associated with the majority of NRPS and PKS gene clusters predicted to produce antimicrobial compounds in the compost-amended FORC inoculated samples. We successfully isolated two \u003cem\u003eStreptomyces\u003c/em\u003e strains from the compost-induced FORC-suppressing cucumber rhizospheres that secrete FORC-inhibiting extracellular metabolites \u003cem\u003ein-vitro.\u003c/em\u003e Their genomes contained NRPS-transAT-PKS and HR-T2PKS-NRPS hybrid clusters that encoded for potential congeners of weishanmycin and ishigamide, respectively. Weishanmycin is a \u003cem\u003eStreptomyces\u003c/em\u003e-produced congener of the well-known, highly potent antitumor drug leinamycin [\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e]. Nonetheless, it may also have antimicrobial attributes, however this along with its native ecological role is currently unknown. Ishigamide is a \u003cem\u003eStreptomyces\u003c/em\u003e derived amide-containing polyene whose function is unknown [\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e]. Polyenes are broad-spectrum antifungal agents produced by \u003cem\u003eStreptomyces\u003c/em\u003e that act by increasing the permeability of the fungal cell membrane through targeting ergosterol, resulting in fungal cell death [\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e]. We posit that these, and potentially additional \u003cem\u003eStreptomyces\u003c/em\u003e-produced compounds could play a crucial role in compost-induced disease suppression through direct fungal antagonism.\u003c/p\u003e \u003cp\u003eIt is imperative to emphasize that our findings represent a specific compost in a specific pathosystem and may not be universal. Future studies should emphasize the potential role of fungi in compost-induced disease suppression and investigate the effects of different composts on rhizosphere microbial communities and functions, as well as their role in disease suppression across various pathosystems. Additionally, designing consortia of strains maintained in eubiosis by compost could help determine their ability to promote disease suppression under disease-conducive conditions. Beyond the production of antimicrobial secondary metabolites, other compost suppressive mechanisms, such as the production of fungal lytic enzymes and activation of plant-induced systemic resistance (ISR), also merit attention.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThis study underlines a link between compost, eubiosis of the rhizosphere microbiome, and mitigation of soilborne pathogen facilitated plant disease, which appears to be analogous to prebiotic-facilitated eubiosis of the gut microbiome that prevents disease in mammals. We posit that compost facilitates a buffering effect that protects the rhizosphere microbiome from pathogen-induced dysbiosis, sustaining \u003cem\u003eActinomycetota\u003c/em\u003e and \u003cem\u003eBacillota\u003c/em\u003e associated strains that potentially antagonize Fusarium through a range of mechanisms including the production of antimicrobial secondary metabolites and siderophores. These findings can be exploited to promote sustainable crop protection strategies that preserve beneficial rhizosphere microbiomes, and to isolate pathogen-antagonizing strains (and their metabolites) that can be as sustainable alternatives to chemical pesticides.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Elad Cohen for his support with computational resources.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors’ contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHA and EC conceptualized and designed the experiments. HA performed the experiments and data analyses. ADF performed shotgun metagenome assembly. \u0026nbsp;HA, JF and EC wrote the manuscript. All authors contributed to the article and approved the submitted version.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was funded by Israel Ministry of Agriculture and Rural Development Chief Scientist grant 20-03-0061.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe raw sequencing data have been deposited in the NCBI Sequence Read Archive (SRA) database under Bioproject PRJNA1169419.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor details\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEC\u003c/strong\u003e-Institute of Soil, Water and Environmental Sciences, Agricultural Research Organization, Rishon Lezion, Israel.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eJF\u003c/strong\u003e-The Institute of Environmental Sciences, The Hebrew University of Jerusalem, Rehovot, Israel.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHA\u003c/strong\u003e- Institute of Soil, Water and Environmental Sciences, Volcani Institute, Agricultural Research Organization, Rishon-Lezion, Israel: Department of Plant Pathology and Microbiology, The Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, Rehovot, Israel.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eADF\u003c/strong\u003e- Department of Vegetable and Field Crops, Institute of Plant Sciences, Agricultural Research Organization, The Volcani Center, Rishon LeZion, Israel.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBonanomi G, Antignani V, Pane C, Scala F. suppression of soilborne fungal diseases with organic amendments. 2007;89:311\u0026ndash;24. \u003c/li\u003e\n\u003cli\u003eFones HN, Bebber DP, Chaloner TM, Kay WT, Steinberg G, Gurr SJ. Threats to global food security from emerging fungal and oomycete crop pathogens. Nat Food. 2020;1:332\u0026ndash;42. http://dx.doi.org/10.1038/s43016-020-0075-0.\u003c/li\u003e\n\u003cli\u003eMihajlovic M, Rekanovic E, Hrustic J, Grahovac M, Tanovic B. Methods for management of soilborne plant pathogens. 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ChemBioChem. 2016;1464\u0026ndash;71. \u003c/li\u003e\n\u003cli\u003eHaro-Reyes T, D\u0026iacute;az-Peralta L, Galv\u0026aacute;n-Hern\u0026aacute;ndez A, Rodr\u0026iacute;guez-L\u0026oacute;pez A, Rodr\u0026iacute;guez-Fragoso L, Ortega-Blake I. Polyene Antibiotics Physical Chemistry and Their Effect on Lipid Membranes; Impacting Biological Processes and Medical Applications. Membranes (Basel). 2022;12. \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"environmental-microbiome","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"sigs","sideBox":"Learn more about [Environmental Microbiome](https://environmentalmicrobiome.biomedcentral.com)","snPcode":"40793","submissionUrl":"https://submission.nature.com/new-submission/40793/3","title":"Environmental Microbiome","twitterHandle":"@bmc","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Rhizosphere, microbiome, FORC, compost, suppressiveness, NRPS, PKS, amplicon sequencing, shotgun metagenomics","lastPublishedDoi":"10.21203/rs.3.rs-5349913/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5349913/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eSoilborne fungal phytopathogens pose a significant threat to global food security. While chemical control remains an effective method for managing these pathogens, increasing regulations due to health and environmental concerns, along with rising fungicide resistance, have restricted their use, underscoring the urgent need for sustainable alternatives. The use of compost to enhance soil fertility and suppress plant diseases is well documented. Several studies have underlined the role of microorganisms in disease suppression, but the mechanisms facilitating this disease suppression remain unclear. We evaluated the impact of compost amendment on the composition and functional capacity of the rhizosphere microbiome in cucumber plants (\u003cem\u003eCucumis sativus\u003c/em\u003e) inoculated with \u003cem\u003eFusarium oxysporum\u003c/em\u003e f. sp. \u003cem\u003eradicis-cucumerinum\u003c/em\u003e (FORC) under controlled greenhouse conditions using amplicon sequencing, shotgun metagenomic and culture-based techniques.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eCompost amendment significantly reduced FORC-induced disease in cucumber relative to non-amended treatments. While FORC inoculation resulted in significant shifts in microbial (bacterial and fungal) community composition in the rhizosphere of non-amended plant, this phenomenon was substantially less pronounced in the rhizosphere of compost-amended plants. Specifically, compost amendment sustained the presence of \u003cem\u003eActinomycetota\u003c/em\u003e (\u003cem\u003eStreptomyces\u003c/em\u003e, \u003cem\u003eActinomadura\u003c/em\u003e, \u003cem\u003eSaccharomonospora\u003c/em\u003e, \u003cem\u003ePseudonocardia\u003c/em\u003e, \u003cem\u003eGlycomyces\u003c/em\u003e, \u003cem\u003eThermobifida\u003c/em\u003e) and \u003cem\u003eBacillota\u003c/em\u003e (\u003cem\u003ePlanifilum\u003c/em\u003e, \u003cem\u003eNovibacillus\u003c/em\u003e) in FORC inoculated plants, that diminished significantly in inoculated plants without compost. These taxa contained a myriad of non-ribosomal peptides (NRPS) and polyketides (PKS) biosynthetic gene clusters (BGCs) with putative antimicrobial and iron-chelating functions. We successfully isolated two \u003cem\u003eStreptomyces\u003c/em\u003e strains from disease suppressed compost amended rhizosphere (almost identical to the most prominent strain identified in the molecular analyses) that produced extracellular metabolites that inhibited growth of FORC \u003cem\u003ein-vitro.\u003c/em\u003e Genome analysis of these strains revealed BGCs that encode for compounds with potential antimicrobial capacity.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eBased on results presented in this study, we demonstrate that compost alleviates FORC-induced dysbiosis of the rhizosphere microbiome, maintaining abundance of specific bacterial taxa. These bacterial groups may contribute to disease suppression through a myriad of mechanisms including iron chelation and production of fungal antagonizing secondary metabolites.\u003c/p\u003e","manuscriptTitle":"Identifying root-associated bacteria and potential mechanisms linked to compost suppressiveness towards Fusarium oxysporum.","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-11-08 14:39:21","doi":"10.21203/rs.3.rs-5349913/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-03-28T14:29:27+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-03-28T01:48:45+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-03-26T21:34:57+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-03-23T11:07:21+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"211057429205103006951270870732054939787","date":"2025-03-23T10:11:07+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"126990917842206297953757367881236579968","date":"2025-03-20T06:52:19+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"177746045309913662569624833531995974637","date":"2025-03-18T22:41:14+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"42283733011239720784578020361262624032","date":"2025-03-18T12:56:09+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"310038255441057595936226143785355719225","date":"2025-03-18T11:14:59+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"221909351790835442207660261796371931655","date":"2025-03-18T07:12:40+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"99162177757271487944919978358956635456","date":"2025-01-21T12:39:42+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"154533780181218422844570835011758495383","date":"2024-11-20T15:45:28+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-11-20T08:09:07+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-11-07T18:44:14+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-10-29T14:03:00+00:00","index":"","fulltext":""},{"type":"submitted","content":"Environmental Microbiome","date":"2024-10-28T22:57:16+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"environmental-microbiome","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"sigs","sideBox":"Learn more about [Environmental Microbiome](https://environmentalmicrobiome.biomedcentral.com)","snPcode":"40793","submissionUrl":"https://submission.nature.com/new-submission/40793/3","title":"Environmental Microbiome","twitterHandle":"@bmc","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"4364ec19-3e0b-4e17-8224-f5585f4fc881","owner":[],"postedDate":"November 8th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-05-19T16:04:33+00:00","versionOfRecord":{"articleIdentity":"rs-5349913","link":"https://doi.org/10.1186/s40793-025-00710-9","journal":{"identity":"environmental-microbiome","isVorOnly":false,"title":"Environmental Microbiome"},"publishedOn":"2025-05-16 15:57:07","publishedOnDateReadable":"May 16th, 2025"},"versionCreatedAt":"2024-11-08 14:39:21","video":"","vorDoi":"10.1186/s40793-025-00710-9","vorDoiUrl":"https://doi.org/10.1186/s40793-025-00710-9","workflowStages":[]},"version":"v1","identity":"rs-5349913","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5349913","identity":"rs-5349913","version":["v1"]},"buildId":"CiT4i_kKBbxQbnFL0ufpk","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}