Healthy wheat plants were collected from Xiping county, Henan Province, in 2012. The roots were cut into sections (1–2 cm long), washed with water for 10 min, surface-sterilized sequentially with 75% ethanol for 30 s and 3% sodium hypochlorite for 1–2 min, and rinsed with sterile distilled water three times, each for 1 min. The sections were dried on a sterile filter paper and placed on potato dextrose agar (PDA: 200 g of peeled potato, 20 g of dextrose, and 20 g of agar in 1,000 mL distilled water) medium containing 150 μg/mL streptomycin and 75 μg/mL penicillin. The root sections were cultured at 25°C for 5–10 days until colonies grew around them; they were then transferred to fresh PDA plates. C. globosum was identified based on the morphologies of the colony, hyphae, and ascospore, as well as the internal transcribed spacer (ITS) and translation elongation factor 1–α (tef1) gene region sequences. Following culture of the isolate on PDA for 7 days, five pieces of marginal hyphae were selected and placed onto a PDA plate covered with sterile cellophane. The hypha was scraped using a small sterile shovel 5 days later, immediately immersed into liquid nitrogen, and stored at −20°C until further analysis. DNA extraction was conducted using the E.Z.N.A.^® HP Fungal DNA Kit (Omega, Norcross, Georgia, United States). The ITS and partial tef1 gene regions were amplified and sequenced by using the primer pair ITS1 (5´-TCCGTAGGTGAACCTGCGG-3′) /ITS4 (5´-TCCTCCGCTTATTGATATGC-3′; White et al., 1990), and EF1-983F (5´-GCYCCYGGHCAYCGTGAYTTYAT-3′) /EF1-2218R (5´-ATGACACCRACRGCRACRGTYTG-3′; S. Rehner, AFTOL, http://aftol.org/), respectively. The PCR products were sequenced by Shanghai Sangon Biological Engineering Co. Ltd.

In our previous study (Xu et al., 2018), the F. pseudograminearum isolate G14LY24–2 was obtained and preserved at the China General Microbiological Culture Collection Center (CGMCC3.20319). G14LY24–2 had strong pathogenicity and was used to conduct dual-culture antagonism assays with C. globosum 12XP1-2-3. Other F. spp. reported in Xu’s study (Xu et al., 2018) that cause wheat crown rot were also used to test the inhibition rates of 12XP1-2-3 (Supplementary Figure 1). Plugs (5 mm in diameter) were collected from the edges of 12XP1-2-3 colonies using a sterile perforator and inoculated onto a PDA plate. After 3 days, plugs of G14LY24–2 and other F. spp. were placed on the same PDA plate at 4-cm intervals with those of 12XP1-2-3. PDA plates inoculated with G14LY24–2 and other F. spp. but without 12XP1-2-3 were used as control. The experiment was repeated three times. The inhibitory effect was observed after culturing at 25°C for 3–7 days and calculated as follows:

Chaetomium globosum 12XP1-2-3 was cultured on PDA plates at 25°C for 10 days. Five plugs (5 mm in diameter) were collected from the edges of 12XP1-2-3 colonies using a sterile perforator and inoculated in a triangular flask containing 100 mL of culture solution. The culture solution was 1% (w/v) glucose, 1% (w/v) sucrose, 0.5% (w/v) beef extract, 0.02% (w/v) iron sulfate, and 0.01% (w/v) vitamin B₁ (pH at 7.0) and was sterilized at 121°C for 30 min (Gao et al., 2010). Flasks containing C. globosum were cultured on an orbital shaker at 25°C and 180 r/min for 28 days. The mycelium was removed via filtration, first using two layers of filter paper in a Buchner funnel connected to a filtering and suction filter, and then using a 0.22-μM sterile microporous membrane.

Fusarium pseudograminearum isolate G14LY24–2 was grown on PDA plates for 3 days at 25°C in the dark. Then 10 pieces of PDA colonized by the fungus (0.25 cm² each) were placed in 100 mL of mung bean liquid medium (4%, w/v), cultured in 250-mL triangular flasks at 25°C on an orbital shaker at 150 rpm for 7 days, and filtered using sterile gauze. The mung bean liquid medium was prepared by boiling 40 g of green beans in distilled water until the pericarp started to crack open; the extract was filtered through several layers of cheesecloth, and the volume was made up to 1L using distilled water. The medium was autoclaved for 20 min at 121°C. The concentration of macroconidia was determined using a hemocytometer, and the suspension was diluted to a final concentration of 1 × 10⁶ spores/mL.

Fermentation broth (50 μL) from 12XP1-2-3 was placed on a sterile concave slide and dried before dropping an equal volume of macroconidia suspension of G14LY24–2 to test for spore germination. A culture solution without 12XP1-2-3 was used as the negative control. The spore germination rate was calculated using a light microscope 6 h after incubation at 25°C, with five visual fields examined for each of the three replicates. For testing on mycelial growth, fermentation broth was mixed with PDA in a volume ratio of 1:4 (culture solution without 12XP1-2-3 was used as control). Each plug (5 mm in diameter) of G14LY24–2 was placed at the center of the above medium. The inhibition rate of growth velocity was calculated by measuring hyphae diameter after 1 and 4 days. The edge of the hyphae was observed using a light microscope.

The control effect of C. globosum ascospores on wheat FCR was investigated both indoors and in the field. C. globosum was inoculated on PDA plates and cultured at 25°C for 15 days. The spore suspension was obtained via surface washing and coating using a glass spreading rod after adding sterile water, followed by filtration using four layers of sterile gauze. The spore suspension (1 × 10⁸ ascospores per milliliter) was mixed with 4% (w/v) sterile sodium carboxymethyl cellulose solution with a volume ratio of 3:1. A 20-mL volume of the mixture was used to coat 1 kg of wheat seeds. By washing the seeds and spread plate, the number of spores on the surface of seeds coated with C. globosum 12XP1-2-3 was determined to be 5.0–5.3 × 10⁴ ascospores per seed. The colonization ability of C. globosum 12XP1-2-3 was tested on wheat seedlings 1 month after sowing. Roots and shoots (1 cm in length) were cut off from 20 seedlings coated with 12XP1-2-3 and surface-sterilized to isolate and identify C. globosum morphologically and molecularly.

Seeds of Triticum aestivum cv. Aikang 58 were prepared and coated with the ascospore suspension of C. globosum 12XP1-2-3 as described in section 2.4. Seeds coated with sterile water and sodium carboxymethyl cellulose solution (with the same ratio of 3:1) were used as negative control (control seeds). The inoculation of wheat seedlings with F. pseudograminearum was conducted indoors according to the method described by Mitter et al. (2006). The macroconidia of F. pseudograminearum isolate G14LY24–2 were prepared as described above and diluted to 1 × 10⁶ spores/mL for seedling inoculation. Twelve wheat seedlings were grown in plastic pots (diameter = 10 cm) containing sterilized soil (50% natural soil and 50% sand, v/v) under glasshouse conditions [12-h photoperiod at a day/night temperature of 25/15°C and relative humidity of 60%/80% (± 5%)]. Ten days after emergence, each seedling was inoculated with a 10-μL droplet of the G14LY24–2 spore suspension at the basal part of the stem, i.e., approximately 0.5–1.5 cm above the surface of the soil. Four treatments were applied, including C. globosum coated seeds plus F. pseudograminearum inoculation (Cg + Fpg), control seeds plus F. pseudograminearum inoculation (Con+Fpg), C. globosum coated seeds without F. pseudograminearum inoculation (Cg–Fpg) and control seeds without F. pseudograminearum inoculation (Con–Fpg). Each treatment included 8 plastic pots (4 pots were used for disease investigation, and the other 4 were used for sampling). The seedlings were incubated at near-saturated relative humidity in darkness for 48 h, then transferred to a glasshouse under the same conditions described above for 35 days. The degree of disease was rated using five grades, namely 0: no disease; 1: trace to 10% of the first leaf sheath discolored; 2: 11–25% of first leaf sheath discolored; 3: 26–50% of the first leaf sheath discolored; 4: ≥ 50% of the first leaf sheath discolored or obviously necrotic second leaf sheath; 5: third leaf sheath obviously necrotic or entire plant severely to completely necrotic. The disease index and control effect were calculated using the following formulas:

Samples of wheat stem bases (2 cm in length) in the four treatment groups were collected 7 and 21 days after inoculation with F. pseudograminearum. There were three replicates in each treatment, with 5–6 stem bases for each replicate. Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, United States), and cDNA was synthesized according to the manufacturer’s instructions. A total of 21 defense-related genes (Supplementary Table 1) were quantified using the SYBR^® Green RT-qPCR kit on a BIO-RAD CFX Connect^™ Real-Time System. These genes were related to antimicrobial defense (PR1, PR2, PR3, PR4, PR5, PR10, and EG), metabolism (PAL1, PAL2, and Glu), signaling (MAP and Stpk-V), ROS defense (TaGLP5 and RP), transcription (WRKY), jasmonic acid pathway (TaAOS, JAZ1, and LIPASE) and so on. The 18S rRNA gene was used as the housekeeping gene for normalization. The reaction system was as follows: 10 μL 2 × qPCR Mix, 2 μL cDNA, 1 μL 10 μmol L⁻¹ of each primer, and 6 μL H₂O. The amplification program was 95°C for 3 min, followed by 40 cycles of 95°C for 10 s, 57°C for 30 s, and 72°C for 35 s. All reactions were performed in triplicate, including three non-template controls. Dissociation curves were generated for each reaction to ensure specific amplification. Relative expression was calculated using 2^–△△Ct. Data were presented as mean with standard error (SE) and analyzed using one-way factorial analysis of variance (ANOVA) and Duncan’s multiple range test (p < 0.05).

We attempted to detect the effect of seed coating treatment of C. globosum (‘Cg’ treatment) on the microbial diversity of the rhizosphere soil of wheat. During the 2019 field investigation in Wenxian, wheat plants (cv. Aikang 58) were pulled up manually with sterile gloves and collected at three growth points, i.e., Feekes 3 (March 1, 2019), Feekes 7 (April 11, 2019) and Feekes 11 (May 23, 2019). Thirty plants from each plot were selected as one of three replicates for each treatment, as described above. Six treatments were applied, defined as Cg.3 (‘Cg’ treatment at Feekes 3), Con.3 (‘Con’ treatment at Feekes 3), Cg.7 (‘Cg’ treatment at Feekes 7), Con.7 (‘Con’ treatment at Feekes 7), Cg.11 (‘Cg’ treatment at Feekes 11), and Con.11 (‘Con’ treatment at Feekes 11). The loose soil around the roots was shaken off, and the thin layer of rhizosphere soil attached to the root surface was collected using a sterile swab, transferred into 2-mL sterile centrifuge tubes, and stored at −70°C (Kobayashi et al., 2015). 16S/ITS amplicon sequencing and analysis were conducted by OE Biotech Co. Ltd. (Shanghai, China). Total genomic DNA was extracted using a MagPure Soil DNA LQ Kit (Magen, Guangdong, China) according to the manufacturer’s instructions. The quality and quantity of DNA were verified by a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, United States) and agarose gel electrophoresis. Extracted DNA was diluted to a concentration of 1 ng/μL and stored at −20°C until further processing. The diluted DNA was used as template for PCR amplification of bacterial 16S rRNA genes with the barcoded primers and Takara Ex Taq^® (Takara). V3–V4 variable regions of 16S rRNA genes were amplified using universal primers 343F and 798R to assess bacterial diversity. ITS I variable regions were amplified using universal primers ITS1F and ITS2 to assess fungal diversity. Amplicons were visualized using gel electrophoresis to determine their quality, purified using AMPure XP beads (Agencourt), and amplified via another round of PCR. The final amplicon was purified again using AMPure XP beads and quantified using a Qubit dsDNA assay kit. Equal amounts of purified amplicon were pooled for subsequent sequencing. Raw sequencing data were generated in FASTQ format. Paired-end reads were then preprocessed using cutadapt software to detect and remove the adapter. After trimming, paired-end reads were filtered, denoised, merged, and detected. Chimera reads were cut off using DADA2 with the default parameters of QIIME2 (2020.11). Lastly, the software generated the representative reads and the ASV abundance table. The representative read of each ASV was selected using QIIME2 package. All representative reads were annotated and blasted against Silva database Version 138 (or Unite; 16 s/ITS rDNA) using q2-feature-classifier with the default parameters.

The differences in the field investigation data, and the alpha index value were analyzed using one-way ANOVA and Duncan’s test (p < 0.05). Beta diversity was analyzed via ADONIS analysis based on Bray–Curtis (bacteria) and Binary-Jaccard (fungi), and visualized by principle coordination analysis (PCoA). The network analysis was designed based on the genus levels of bacteria and fungi in each treatment (‘Cg’ and ‘Con’) at all three stages. Genera with total relative abundances less than 0.5% were discarded. The relationship among genera was examined via Spearman’s correlation using R (version 4.2.1) with the value of p adjusted using the Benjamin Hochberg method. Genera with significant correlations (|r| > 0.8 for bacteria and > 0.7 for fungi, p < 0.001) were visualized using the Fruchterman-Reingold layout in Gephi. LEfSe (Linear discriminant analysis Effect Size) analysis (Galaxy) was applied to identify biomarkers among three stages (LDA > 3), as well as between the ‘Cg’ and ‘Con’ treatments at each stage (LDA > 3 for bacteria and > 2 for fungi). The correlation among the relative abundance of specific biomarkers, as well as fungal Shannon and disease indices, was analyzed via the Spearman method.

The morphological and molecular characteristics of isolate 12XP1-2-3 was in line with the characteristics of C. globosum. It was preserved at the China General Microbiological Culture Collection Center (CGMCC17183). Additionally, the sequences of ITS and tef1 gene regions were uploaded to the NCBI database under accession number OL721626 and OQ378355. The tablet confrontation test (Figures 1A, B) showed that mycelial growth of F. pseudograminearum G14LY24–2 was apparently inhibited in the co-culture group (Figure 1B) compared with that in the control group (Figure 1A). The tablet inhibition rate of 12XP1-2-3 against F. pseudograminearum G14LY24–2 was 65.9%. Its inhibition rates against other F. spp. causing FCR were shown in Supplementary Figure 1 and ranged from 27.9% (F. proliferatum) to 72.0% (F. graminearum and F. asiaticum).

As shown in Figure 1, a higher number of F. pseudograminearum spores burgeoned after 6 h, with longer mycelium in the control group (Control; Figure 1C) than in the fermentation-broth-treated group (Cg; Figure 1D). The spore germination rate in the control group was 94.9%; however, the fermentation-broth-treated group showed a germination rate of only 42.4%, exhibiting an germination inhibition of 55.3%.

The mycelial growth of F. pseudograminearum was inhibited in PDA supplemented with 12XP1-2-3 fermentation broth (Figures 1E,F), and the inhibition rate of growth velocity was 25.4% after 4 days of culture. Microscopic observation showed that the edge of the hyphae in PDA mixed with fermentation broth was abnormal and deformed, with an expanded hyphal tip and markedly more shortened intervals of mycelia branches (Figures 1G,H).

The isolation rates of C. globosum in roots and shoots of seedlings coated with 12XP1-2-3 were 75 and 80%, respectively, indicating that C. globosum 12XP1-2-3 had a high colonization rate on wheat. Seedlings coated with and without C. globosum 12XP1-2-3 showed no significant differences in growth indices. Seven days after inoculation with F. pseudograminearum G14LY24–2, most wheat seedlings in the control group had a brown stem base (Figure 2D), and the percentage of diseased plants was 66.6% (Figure 2A). However, most seedlings coated with 12XP1-2-3 spores (‘Cg + Fpg’ treatment) showed almost no disease symptom (Figure 2C), with only a diseased plant rate of 3.95% (Figure 2A). The disease index of ‘Cg + Fpg’ treatment 35 days after inoculation was 12.23 and was significantly decreased (p < 0.05) from that of ‘Con+Fpg’ (19.5), resulting in a control effect of 37.3% (Figure 2B). Therefore, the occurrence of wheat crown rot may be delayed and the disease index was significantly decreased via the C. globosum (‘Cg’) treatment.

The relative expression of 21 defense-related genes was detected in wheat stem bases 7 and 21 days after inoculation (Figure 2E; Supplementary Figure 2). After 7 days, 12 genes in the ‘Con+Fpg’ treatment showed significantly (p < 0.05) higher relative expression than those in the ‘Cg + Fpg’ treatment, including genes related to antimicrobial defense (PR1, PR2, PR3, PR4, PR5, PR10, and EG), metabolism (PAL1 and PAL2), ROS defense (TaGLP5 and RP) and others (TaWIR1b). Based on the delayed occurrence of FCR through seed coating with C. globosum, we supposed that the up-regulation of these defense-related genes might also be postponed in the ‘Cg’ group. Conversely, most defense-related genes were significantly (p < 0.05) and more highly expressed in the ‘Cg + Fpg’ treatment 21 days after inoculation, as compared with those in the ‘Con + Fpg’ treatment, including genes related to antimicrobial defense (PR1, PR2, PR3, PR4, PR5, and PR10), metabolism (PAL1, PAL2, and Glu), signaling (Stpk-V), ROS defense (TaGLP5 and RP), transcription (WRKY), Jasmonic acid pathway (TaAOS and JAZ1) and others (PDR).

Field trials were conducted in Kaifeng, Wenxian, and Neihuang from 2018 to 2020 using wheat cv. Aikang 58 and Bainong 207. Wheat from the ‘Cg’ treatment group showed better growth traits in every field trial during the whole test period (Supplementary Figure 3). Although there were no significant differences in plant height, root length, fresh weight, and dry weight among the three treatments at Feekes 3 and Feekes 7, the chlorophyll content of flag leaves in the ‘Cg’ treatment group was significantly higher than those in the ‘Dif’ and ‘Con’ treatment groups at Feekes 7 (Supplementary Table 2), demonstrating that seed coating with 12XP1-2-3 might promote wheat growth. Our findings showed that disease control effects of both ‘Cg’ and ‘Dif’ treatments varied in different years/locations/varieties (Table 1). The ‘Dif’ and ‘Cg’ treatments exhibited similar control effects in Kaifeng in 2018. The rates of diseased stems at Feekes 7, as well as rates of diseased stems and disease indices at Feekes 11 in both treatments were all lower than those in the control (‘Con’). Moreover, there were significant differences (p < 0.05) between ‘Cg’ and ‘Con’, as well as between ‘Dif’ and ‘Con’ in the rates of diseased stems and disease indexes at Feekes 11. The disease control effect was 35.6% in the ‘Dif’ treatment and 43.1% in the ‘Cg’ treatment. Both treatments led to increased yield, with increased rates of 1.2% for ‘Dif’ and 3.2% for ‘Cg’. During the two trials at Wenxian in 2019 and Neihuang in 2020, the rates of diseased stems at Feekes 7 and Feekes 11 for both treatments (‘Dif’ and ‘Cg’) were lower than those of the control (‘Con’); however, no significant differences were observed (p > 0.05). The disease indices of ‘Dif’ and ‘Cg’ treatments at Feekes 11 showed significant differences compared with those of the control (‘Con’). The disease control effects of ‘Dif’ and ‘Cg’ treatments at Wenxian in 2019 were 70.5 and 45.0%, respectively, and the increased rates of yield were as high as 10.7 and 11.9%, respectively. However, the disease control effects of ‘Dif’ and ‘Cg’ treatments at Neihuang in 2020 were only 28.4 and 25.9%, respectively. The ‘Cg’ treatment resulted in increased yield at a rate of 5.9%; however, the yield of wheat in the ‘Dif’ treatment (404.83 kg/667 m²) was lower than that of the control (424.02 kg/667 m²). At Wenxian in 2020, the ‘Cg’ treatment resulted in the highest disease control effect (73.1%) and an increased rate of yield at 8.8%; however, the ‘Dif’ treatment showed more serious disease progression and reduced yield compared with the control.

The numbers of valid bacterial tags were between 52,201–57,585, and the numbers of amplicon sequence variants (ASVs) ranged from 993–1,660 for all samples. The numbers of valid fungal tags were between 62,549–69,669, and the numbers of ASVs ranged from 157–321. The Shannon index dilution curve in each group was gentle (Supplementary Figure 4), demonstrating that the sequencing depth had covered most species. The data volume adequately reflected the diversity of species in each sample.

The richness (Chao 1 index) and species diversity (Shannon and Simpson indices) of rhizosphere microorganisms were calculated (Figure 3). The Chao 1, Shannon, and Simpson indices of the bacterial community in the control group all increased gradually during the three periods and were highest at Feekes 11 (Chao 1: 1,231 < 1,340 < 1,573; Shannon: 8.33 < 9.05 < 9.68; Simpson: 0.982 < 0.994 < 0.998). Significant differences (p < 0.05) were observed between the Chao 1 indices at Feekes 7 and Feekes 11, the Shannon indices among the three stages, and the Simpson indices at Feekes 3 and Feekes 7. The Chao 1, Shannon, and Simpson indices of the fungal community in the control group (‘Con’) first increased, then decreased, during the three stages (Chao 1: 226.95 < 284.09 > 220.76; Shannon: 4.98 < 5.30 > 4.03; Simpson: 0.909 < 0.930 > 0.804); however, no significant differences were observed (p > 0.05). The Chao 1, Shannon, and Simpson indices of the bacterial communities in the ‘Cg’ and ‘Con’ groups showed no significant differences at each growth stage, indicating that the ‘Cg’ treatment had no significant influence on richness and species diversity of the rhizosphere bacteria during the growth period of wheat. The fungal Chao 1, Shannon, and Simpson indices of the ‘Cg’ treatment were lower (Chao1: 182.57 and 267.33; Shannon: 3.90 and 5.07; Simpson: 0.825 and 0.908) than those of the control at Feekes 3 and Feekes 7, with no significant differences. However, they increased at Feekes 11 (Chao 1: 248.37; Shannon: 5.11; Simpson: 0.933) compared with the control. Moreover, significant differences were observed between the Shannon indices at Feekes 11 (pairwise comparison, p = 0.022). These results indicated that the ‘Cg’ treatment had no significant influence on richness and species diversity of the rhizosphere fungi at Feekes 3 and Feekes 7 but significantly increased the Shannon index at Feekes 11.

PCoA showed that the effect of growth periods on microbial clustering was greater than that of the seed dressing treatments (Figures 4C,D). The separations among the ‘Cg’ and control treatments at each growth stage were not obvious in both bacterial and fungal communities. Both the bacterial and fungal communities were divided during the three growth periods, which could explain 39.55% (PC1) and 15.19% (PC2) of total variations for bacteria (Figure 4C), and 13.49% (PC1) and 10.44% (PC2) of total variations for fungi (Figure 4D).

A total of 33 phyla, 76 classes, 196 orders, 303 families and 586 genera from the bacterial community were identified among all groups. The top 10 most abundant genera were Massilia, Flavobacterium, Sphingomonas, Nocardioides, Lysobacter, Pseudomonas, Pseudarthrobacter, Promicromonospora, Devosia, and Pedobacter (Figure 4A). During the three growth stages, Massilia (at Feekes 3), Pseudomonas (at Feekes 3), and Promicromonospora (at Feekes 7) were the biomarkers for the control treatments (Con.3, Con.7, and Con.11) according to LEfSe analysis with LDA > 3 (Figure 5A). The relative abundance of Massilia and Pseudomonas decreased gradually in both ‘Cg’ and ‘Con’ groups during the three growth stages, with 15.43–17.32% and 4.10–4.76% at Feekes 3, 6.52–6.96% and 2.78–3.05% at Feekes 7, and 1.52–1.96% and 0.87–0.94%, at Feekes 11, respectively. The relative abundance of Promicromonospora was 1.38–2.09% at Feekes 3, 3.34–4.76% at Feekes 7, and 0.83–0.85% at Feekes 11.

A total of 9 phyla, 25 classes, 62 orders, 102 families and 151 genera from the fungal community were identified in all groups. The top 10 most abundant genera were Fusarium, Monographella, Mortierella, Olpidium, Sarocladium, Cortinarius, Penicillium, Thanatephorus, Cladosporium, and Cystofilobasidium (Figure 4B). Six of these genera were the biomarkers during three growth stages for the control treatments (Con.3, Con.7, and Con.11) according to LEfSe analysis (LDA > 3), including Mortierella at Feekes 3, Thanatephorus and Cystofilobasidium at Feekes 7, and Fusarium, Sarocladium, and Cladosporium at Feekes 11 (Figure 5B). Cystofilobasidium was reported to be a type of biocontrol fungi, whereas the other five genera were known as plant pathogens. The relative abundance of Fusarium in the ‘Cg’ and ‘Con’ treatment groups increased gradually as the wheat grew (4.39–4.44% at Feekes 3, 5.98–10.85% at Feekes 7, and 12.09–39.44% at Feekes 11). The relative abundance of Sarocladium and Cladosporium also showed a similar increasing trend. However, that of Mortierella exhibited an opposite pattern, i.e., relative abundance decreased gradually over time and was 4.72–12.97% at Feekes 3, 3.98–4.21% at Feekes 7, and 1.91–2.50% at Feekes 11. The relative abundance of Thanatephorus and Cystofilobasidium increased first and then decreased.

Bacterial networks showed more edges, as well as a higher negative edge rate, clustering coefficient, average degree, and network density in the ‘Cg’ treatment group than in the control group. Conversely, fungal networks in the ‘Cg’ treatment exhibited fewer nodes and edges, as well as a lower negative edge rate, clustering coefficient, average degree, and network density than the control (Figures 6A–D; Supplementary Table 3). Furthermore, the top 10 hub nodes of the ‘Cg’ bacterial network displayed a higher degree and closeness centrality than those of the control, but those of the ‘Cg’ fungal network were on the contrary (Figures 6E–H). The negative edge rate of the ‘Cg’ bacterial network (38.2%) was higher than that of the control (31.3%) but was reduced in the ‘Cg’ fungal (27.4%) network compared with the ‘Con’ fungal (39.0%) network (Figure 6I; Supplementary Table 3). These results demonstrated that the ‘Cg’ bacterial network exhibited more complexity and stability than the control. These characteristics of the co-occurrence networks were very similar to the results reported by Yuan et al. (2020) and Gao et al. (2021), who discussed that the healthy bacterial network showed higher complexity than the diseased network, whereas the fungal network showed a contrasting pattern. Thus, it can be concluded that both the bacterial and fungal ‘Cg’ networks are closer to the healthy state, whereas the ‘Con’ networks are closer to the diseased state. Therefore, the ‘Cg’ treatment may promote the health degree of the wheat rhizosphere microbial network.

We further explored the effect of seed coating by C. globosum on the microbial community. The ‘Cg’ groups in every growth stage were compared with their corresponding ‘Con’ groups through the LEfSe analysis. As shown in Figure 5C, LEfSe identified Bacillus (0.76 vs. 0.47%), Rhizobium (including Allorhizobium, Neorhizobium, Pararhizobium, and Rhizobium; 1.24 vs. 0.87%), Duganella and Pantoea as biomarkers in the Cg.3 group when compared with the Con.3 group (LDA > 3), indicating that the relative abundances of beneficial bacteria such as Bacillus and Rhizobium increased in the ‘Cg’ treatment at Feekes 3. Sphingomonas (5.22 vs. 4.36%) was one of the biomarkers in the Cg.7 group (compared with the control), which was reported as a new microbial resource to degrade aromatic compounds.

The results obtained for the fungal community are shown in Figure 5D. LEfSe results from the Cg.3 vs. Con.3 combination indicated that, at Feekes 3, the relative abundances of pathogens Fusarium (4.44 vs. 4.39%) and Paramyrothecium (0.44 vs. 0.45%) were not significantly different between the ‘Cg’ and ‘Con’ treatments. At Feekes 7, the relative abundance of Fusarium in the ‘Cg’ treatment (Cg.7, 5.98%) was lower than that in the control (Con.7, 10.85%), but with no significant differences. The relative abundance of the pathogen Stemphylium significantly increased (0.29 vs. 0.05%) in the ‘Cg’ treatment compared with the control. At Feekes 11, the relative abundance of Fusarium significantly decreased (12.09 vs. 39.44%), whereas that of Monographella significantly increased (7.29 vs. 3.81%) in the ‘Cg’ treatment compared with the control.

We performed Spearman correlation analysis to determine the correlation among the relative abundance of the above important biomarkers, fungal shannon index and disease index (Figure 5E). The results revealed that Bacillus at Feekes 3 had a significant negative correlation with the disease index and relative abundance of Fusarium at Feekes 11 but positive with the fungal Shannon index at Feekes 11. Relative abundance of Rhizobium at Feekes 3 had a positive correlation with that of Sphingomonas at Feekes 7. Relative abundance of Sphingomonas at Feekes 7 had a negative correlation with that of Fusarium at Feekes 11. Relative abundance of Fusarium at Feekes 11 had a negative correlation with the fungal Shannon index and a positive correlation with the disease index, both at Feekes 11. Furthermore, the fungal Shannon index at Feekes 11 negatively correlated with the disease index. Therefore, the integrative action of Bacillus, Rhizobium, and Sphingomonas during the early growth period (Feekes 3 and 7) probably contributed to Fusarium relative abundance, the fungal Shannon index, and the disease index at Feekes 11.