Development of biocontrol agents for cotton verticillium wilt using microbiome analysis

Development of biocontrol agents for cotton verticillium wilt using microbiome analysis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Development of biocontrol agents for cotton verticillium wilt using microbiome analysis Wanyi Zhang, Jingjing Wang, Peng Wu, Yan Pang, Shijia Dai, Xiaoxia Zhang, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6884856/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Verticillium wilt is one of the most devastating diseases of cotton. However, effective biocontrol strains are still lacking. The aim of this study is to inform the selection of effective biocontrol strains by comparing the microbiomes of healthy and diseased cotton plants. Our results revealed that Verticillium dahliae V991 (V991) is the causal agent of cotton Verticillium wilt, significantly altering the bacterial and fungal communities in the roots, rhizosphere and bulk soil. Compared to the diseased cotton in the V991 inoculation group (D), the healthy cotton in the V991 inoculation group (H) and the control cotton in the V991 non-inoculation group (C) both suppressed Verticillium and Fusarium and enriched taxa of Bacilli , Clostridia , Archacosporales , Glomerales , unclassified Basidiomycota and unclassified Glomeromycota in the roots, both enriched Burkholderiales in the rhizosphere soil, both enriched Archaeosporales and Verrucomicrobiota in the bulk soil. A total of 20 strains were screened for antagonism to V991, most of them were isolated from the roots of the C group. Bacillus amyloliquefaciens M9 (BM), which was screened from the rhizosphere soil, exhibited the strongest antifungal activity, whereas Bacillus cereus R19 (BR), which was screened from the root, exhibited weaker antifungal activity. Pot experiments showed that the application of BR and BM (10 9 CFU/mL) reduced the disease incidence by 44.44% and 33.33%, respectively, compared to the control. Field experiments showed that BR reduced the disease incidence by 88.46%, while BM reduced it by 50.01%. These results demonstrate the effectiveness of comparative microbiome analysis in guiding the selection of highly effective biocontrol strains. cotton verticillium wilt microbial communities bio-control bacteria antifungal activity Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction China is the world’s largest consumer and second-largest producer of cotton (Feng et al., 2017 ). The quality and yield of cotton are severely affected by Verticillium wilt, a soil-borne disease caused by the Verticillium dahliae pathogen (Zhu et al., 2023 , Yang et al., 2025 ). Unfortunately, Verticillium wilt is currently considered the ‘cancer of cotton’. It generally causes plant dysplasia, wilting and yellowing of leaves, and browning of vascular bundles, and eventually leading to early death (Zhang et al., 2022 ). In recent years, the incidence of Verticillium wilt has increased, resulting in significant economic losses in the cotton industry (Zhang et al., 2025 ). Controlling the wide spread of V. dahliae remains huge challenge due to its stable dormant microsclerotia structure, long-term variability and coevolution with host plants (Kong et al., 2022 ). Although soil fumigation, chemical pesticides and crop rotation are common methods of controlling Verticillium wilt, they cause significant environmental damage and only alleviate diseases to a limited extent, making them unsuitable for large-scale application (Carroll et al., 2018 ). Biological control, which leverages antagonistic microorganisms to suppress pathogens, offers a sustainable alternative to chemical interventions (Berg, 2009 ). This approach aligns with global efforts to reduce pesticide use and promote eco-friendly agriculture. In the case of cotton Verticillium wilt, potential biocontrol agents include bacteria (e.g., Bacillus spp.) (Zhou et al., 2021 , Song et al., 2024 ) and non-pathogenic isolates of Fusarium (Zhang et al., 2024 ). These organisms employ various mechanisms to inhibit V. dahliae , such as competing for resources and producing antifungal metabolites (e.g., lipopeptides, siderophores), as well as inducing systemic resistance in plants. However, effective biocontrol strains for field application are still lacking. Advances in high-throughput sequencing and microbiome analysis have revolutionized our understanding of plant-microbe interactions. The soil and root microbiome plays a pivotal role in plant health and agricultural sustainability. These microorganisms form intricate symbiotic relationships with plants, contributing to nutrient acquisition, stress resilience, and pathogen suppression (Trivedi et al., 2020 ). Comparative microbiome analysis, a powerful tool for identifying microbial taxa associated with disease resistance, has become an increasingly popular method for guiding the selection of biocontrol strains. Mendes et al. ( 2011 ) developed a framework for using comparative microbiome analysis to prioritize candidate biocontrol agents. They compared rhizosphere microbiomes from disease-suppressive and conducive soils and identified γ- Proteobacteria , especially Pseudomonadaceae , as key drivers of suppression to Rhizoctonia solani . They then isolated Pseudomonas from suppressive soils and found that it could suppress Rhizoctonia solani in vitro and in greenhouse trials. Kwak et al. ( 2018 ) conducted comparative analyses of rhizosphere microbiomes from resistant and susceptible tomato enabled the identification and assembly of a flavobacterial genome that was far more abundant in the resistant plant rhizosphere microbiome than in that of the susceptible plant. They cultivated this flavobacterium, named TRM1, and found that it could suppress Ralstonia solanacearum disease development in a susceptible plant in pot experiments. Zeng et al. ( 2022 ) investigated the endophytic microbiome of healthy and Verticillium wilt infected cotton plants, founding that Pseudomonas was significantly depleted in the diseased plants. They then isolated Pseudomonas from the healthy cotton plants and demonstrated that plants inoculated with Pseudomonas sp. strains showed increased resistance to the cotton Verticillium wilt compared with the control plants in pot experiments. However, there is still a lack of comparative microbiome analysis across multiple sites. In this paper, we first investigated whether Verticillium dahliae V991 causes cotton Verticillium wilt using pot experiments. We then analyzed the microbial communities in the roots, rhizosphere and bulk soil of healthy and diseased cotton plants using amplicon sequencing. Based on these results, we targeted screening biocontrol strains against V. dahliae 991and tested the ability of typical strains to control Verticillium wilt in pot and field experiments. Materials and Methods Strains The Verticillium dahliae V991 strain (National Center for Biotechnology Information (NCBI) accession number PV450007) and Fusarium oxysporum F679 strain (NCBI accession number PV450006) were stored at the Applied Microbial Ecology Engineering Lab at Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences. Pathogenic experiment of V. dahliae V991 The pathogenicity of V. dahliae V991 was tested using a cotton pot experiment. The cotton seeds were sterilized with 3% sodium hypochlorite solution for 15 min. The surface-sterilized seeds were germinated in water for 48 h before being sown in nursery substrate (peaty soil: forest humus: vermiculite: perlite ( v : v ) = 2:1:1:1) at the cotyledon stage (day 5–7). V. dahliae V991 was cultured in PDA medium (Beijing Solarbio Science & Technology Co., Ltd.) at 30℃ for 5 days. The concentration of fungal spores was adjusted to 10 5 CFU/mL using sterile water. On day 7, the 1 cm root tips of cotton seedlings were cut off and soaked in the V. dahliae V991 spore solution (V991) or sterile water (CK) for 3 min. Each treatment consisted of 30 cotton plants. The pots were then placed randomly in a greenhouse with a relative humidity of 70–80% and 16 h of light. Before seedling emergence, the temperature was maintained at 30℃. Thereafter, the temperature was maintained at 28℃ for all subsequent stages. The pots were watered with 500 mL every 48 h and their position was changed randomly. The disease index and incidence rate were recorded in cotton plants on the 25th day after infection by V. dahliae V991. Disease indices were recorded according to the following criteria: Grade 0, healthy strain with no obvious pathological symptoms. Grade 1, 1–2 cotyledons show pathological symptoms. Grade 2, 1–2 true leaves show obvious pathological symptoms. Grade 3, 3 or more true leaves show pathological symptoms or only the heart leaf fall. Grade 4, all leaves fall, the growing point dies or the whole plant dies [30] . Microbial diversity analysis Two healthy and two diseased cotton seedlings from the V991 inoculation (V991) were taken as the healthy group (H) and diseased group (D). Two cotton seedlings from the non-inoculation (CK) were taken as control group (C). On day 25 after infection by V991, bulk soil, rhizosphere soil and roots from these three groups (H, D and C) were collected to analyze the microbial structure (Fig. 1 a). The cotton seedlings were carefully taken from the nursery substrate. Bulk soil, which was loosely attached to the roots, was collected by shaking. The rhizosphere soil, which was adhering to the roots, was collected by washing with 25 mL sterile water and then centrifuging at 2500 g for 5 min. The roots were sterilized by soaking them in 1% sodium hypochlorite solution for 10 min followed by washing them 3–4 times with sterile water. The roots were then soaked in 75% ethanol for 5 min, after which they were rinsed extensively with sterile water a further 3–4 times. The sterilized roots were collected as root samples. There were four samples in each group. All samples were immediately stored at -80°C for further analysis. Information on the microbial community was obtained by performing DNA extraction and sequencing of the samples at Biomarker Technologies Co., Ltd (Beijing, China). All primer pairs provided by the company for sequencing are listed in Table 1 . The raw sequence data were deposited in the NCBI Sequence Read Archive under the accession number PRJNA1071027. The raw data were processed and analyzed using BMKCloud ( www.biocloud.net ). Table 1 The primers used to amplify bacterial and fungal communities Target microorganism Primer Sequence Endogenous bacteria 335F 5'-CADACTCCTACGGGAGGC-3' 769R 5'-ATCCTGTTTGMTMCCCVCRC-3' Endogenous fungi ITS1F 5'-CTTGGTCATTTAGAGGAAGTAA-3' ITS2-Fungal-ad 5'-TGCGTTCTTCATCGATGC-3' Bacteria 515F 5'-GTGYCAGCMGCCGCGGTAA-3' 926R 5'-CCGYCAATTYMTTTRAGTTT-3' Fungi ITS1F 5'-CTTGGTCATTTAGAGGAAGTAA-3' ITS2 5'-GCTGCGTTCTTCATCGATGC-3' ------------------------------------------- Table 1 ------------------------------------------- Isolation and identification of bacteria Bacteria were isolated from the roots, rhizosphere soil and bulk soil of cotton plants. Suspensions of the rhizosphere soil and bulk soils were diluted to 10 − 3 and 10 − 4 CFU/mL, respectively. Then 100 µL of each dilution was plated onto Luria-Bertani (LB) medium containing 2.5 µg/mL amphotericin B. The roots were crushed and shaken with 1 mL sterile water. Then 100 µL of the resulting suspensions was plated onto LB containing 2.5 µg/mL amphotericin B. All colonies were picked from the plates and further re-streaked on fresh LB plates for purification. After 48 h of incubation at 30℃, the isolates were selected based on the morphology (colour and size) of the bacterial colonies for 16S rRNA gene sequencing and identification. Genomic DNA was extracted from the bacterial monocultures using a TIANamp Bacteria DNA Kit (TIANGEN Biotech Co., Ltd. Beijing, China) following the manufacturer’s instructions. PCR amplification of the 16S rRNA gene was performed using the forward primer 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and the reverse primer 1492R (5'-TACGACTTAACCCCAATCGC-3') [31] . The PCR cycle consisted of an initial denaturation at 95℃ for 3 min, followed by 34 cycles of denaturation at 95℃ for 30 s, denaturation at 55℃ for 30 s, and extension at 72℃ for 90 s and a final extension at 72℃for 5 min. The PCR products were sequenced by Tsingke Biotechnology Co. Ltd. company (Beijing, China). All bacterial isolates were identified using the BLAST search program and the NCBI database. Highly homologous sequences were aligned and isolates with the matching genes that were more than 97% identical were considered to be the same species. All strains were stored in 25% glycerol solutions and placed in an ultra-low temperature refrigerator at -80℃. Antifungal activity of the isolated bacteria The antagonistic activity of each isolated bacterial strain against V. dahliae V991 or Fusarium oxysporum F679 ( F. oxysporum F679) was evaluated using the inhibition zone method. A single bacterial colony was inoculated into 1 mL of sterile water to adjust the bacterial concentration to 10 9 CFU/mL. Then, 5 µL of the bacterial solution was added to the PDA medium containing the spread pathogens (10 5 CFU/mL) after 1 h. The plates were sealed with parafilm and incubated for 5 days at 26 ℃. Each treatment included 3 replicates. On day 5, the diameter of the inhibition zone formed by the bacteria growing on the PDA medium with pathogens was measured. The larger the inhibition zone, the stronger the bacteria’s antifungal ability. Biocontrol activity in pot experiments For pot experiments, we selected the strongest V991 antagonist, B. amyloliquefaciens M9 (BM), and the weaker B. cereus R19 (BR), to verify the difference between in vitro and in vivo antagonism. The bacteria were cultured in LB liquid medium (30℃, 180 rpm) for 48 h, and then diluted to different concentrations (10 7 , 10 8 and 10 9 CFU/mL). The cotton seeds were sterilized with 3% sodium hypochlorite solution for 15 min, after which they were soaked in water for 48 h to accelerate germination. The pot experiments comprised 7 treatments involving germinated cotton seeds that were soaked in B. amyloliquefaciens M9 (BM) and B. cereus R19 (BR) at concentrations of 10 7 , 10 8 and 10 9 CFU/mL, respectively (Table 2 ) and in sterile water (CK). Each treatment included 36 seedlings. The germinated cotton seeds of the BM7, BM8 and BM9 were soaked in a B. amyloliquefaciens M9 (BM) solution at concentrations of 10 7 , 10 8 and 10 9 CFU/mL, respectively. The germinated cotton seeds of the BR7, BR8 and BR9 were soaked in a B. cereus R19 (BR) solution at concentrations of 10 7 , 10 8 and 10 9 CFU/mL, respectively. All seeds were soaked for 5 h and then sown in the nursery substrate (peaty soil: forest humus: vermiculite: perlite ( v : v : v : v ) = 2:1:1:1). Table 2 The treatments in pot experiments Treatments Inoculants Concentration CK Sterile water / BM7 B. amyloliquefaciens M9 10 7 CFU/mL BM8 B. amyloliquefaciens M9 10 8 CFU/mL BM9 B. amyloliquefaciens M9 10 9 CFU/mL BR7 B. cereus R19 10 7 CFU/mL BR8 B. cereus R19 10 8 CFU/mL BR9 B. cereus R19 10 9 CFU/mL ------------------------------------------- Table 2 ------------------------------------------- On day 10, the cotton seedlings, which had two unfolded cotyledons, were carefully removed from the nursery substrate. The root tips of all the cotton seedlings were cut to 1 cm, and the wounded roots were infected with 100 mL of V991 (10 5 CFU/mL). On day 25, plants from each pot were harvested, carefully separated into roots and shoots after gentle washing with tap water, and growth parameters, including disease incidence rate, root length and plant height and dry weight were determined. The lower disease incidence rate indicated the stronger biocontrol ability of the tested bacteria. All statistical tests performed in this study were considered significant at P < 0.05. Biocontrol activity in field experiments The biocontrol activity of B. amyloliquefaciens M9 (BM) and B. cereus R19 (BR) was tested in field experiments. The bacteria were cultured in LB liquid medium at 30℃, 180 rpm for 48 h, resulting in a final concentrations of approximately 1×10 10 CFU/mL. The field experiments were conducted in Beiquan Town, Shihezi City, Xinjiang, China. Three treatments were carried out, CK (inoculation with water), BM (inoculation with B. amyloliquefaciens M9) and BR (inoculation with B. cereus R19). Three plots were established for each treatment. Each plot was 4 m 2 in size. One month after sowing the cotton, 30 mL of microbial inoculant or water was applied to each plot. During the cotton harvest, 10 cotton plants were randomly selected from each plot and the incidence rate of Verticillum wilt was counted using the pole-throwing method for each treatment. Results Inoculation with V. dahliae V991 caused severe verticillium wilt in cotton Ten days after inoculation with V. dahliae V991 (V991), the cotton leaves wilted and the roots and stems showed signs of black rot (Fig. 1 a). After 25 days, the disease incidence rate in the cotton seedlings reached 90%, with an incidence index of 77.5 (Fig. 1 b-c). In contrast, all cotton seedlings in the non-inoculation group (CK) remained healthy. These results strongly suggest that V. dahliae V991 is the causal agent of Verticillium wilt in cotton. ------------------------------------------- Fig. 1 ------------------------------------------- Inoculation with V. dahliae V991 altered microbial diversity Interestingly, two of the cotton seedlings in the V. dahliae V991 inoculation (V991) exhibited resistance to the disease. These two cotton seedlings were collected to form the healthy group (H). Two cotton seedlings from the non-inoculation group (CK) were then collected as the control group (C) and two from the V. dahliae V991 inoculation (V991) as the disease group (D) (Fig. 1 a). Bulk soil, rhizosphere soil and roots from these three groups (H, D and C) were collected on day 25 after infection by V. dahliae V991 to analyze the microbial structure. The results showed that the effect of location on microbial diversity far outweighs the effect of disease severity (Fig. S1 ). Bacterial and fungal α diversity (Shannon index) was lower in the disease group (D) than in the healthy group (H) and the control group (C) (Fig. 2 a-f). However, only endogenous bacterial α diversity was significantly lower in the disease group (D) compared to the healthy group (H) and the control group (C) (Fig. 2 a). Endogenous fungal α diversity was significantly lower in the control group (C) than in the healthy group (H) (Fig. 2 d). No significant differences were observed in the α diversity of bacteria and fungi in the rhizosphere and bulk soil among the three groups (Fig. 2 b-c and Fig. 2 e-f). Significant differences in the β diversity of the bacterial and fungal communities were observed in the root, rhizosphere soil and bulk soils (Fig. 2 g-l). The difference in bacterial communities in bulk soil was obviously greater than in the root and rhizosphere soil (Fig. 2 g-i). The difference in fungal communities from the roots was obviously greater than that from the rhizosphere soil and bulk soils (Fig. 2 j-l). The differences in bacterial and fungal communities between the diseased and control groups (D-C) and between the diseased and healthy groups (D-H) were greater than those between the healthy and control groups (H-C) (Table 3 ). These results suggest that infection with V. dahliae V991 altered the diversity of the fungal and bacterial communities. Table 3 Permutational multivariate analysis of variance (PERMANOVA) of microbial β diversity Bacteria Fungi R 2 P -value R 2 P -value Root (E) C-H 0.149 0.031 0.270 0.098 C-D 0.157 0.046 0.494 0.001 H-D 0.159 0.023 0.504 0.027 Rhizosphere soil (R) C-H 0.189 0.087 0.206 0.027 C-D 0.250 0.030 0.217 0.001 H-D 0.252 0.032 0.207 0.001 Bulk soil (S) C-H 0.157 0.030 0.159 0.038 C-D 0.249 0.001 0.183 0.001 H-D 0.330 0.001 0.190 0.032 C, control group; H, healthy group; D, diseased group. ------------------------------------------- Fig. 2 ------------------------------------------- ------------------------------------------- Table 3 ------------------------------------------- Inoculation with V. dahliae V991 altered the microbial composition The dominant bacteria in the root of the control group (C) and the healthy group (H) were Proteobacteria and Firmicutes at the phylum level (Fig. S2 a), and uncultured_Mollicutes_bacterium and Candidatus_Solibacter at the genus level (Fig. 3 a). In the diseased group (D), the dominant bacteria in the roots were Proteobacteria and Myxococcota at the phylum level (Fig. S2 a), and Allorhizobium _ Neorhizobium _ Pararhizobium _ Rhizobium and Pseudomonas at the genus level (Fig. 3 a). The dominant bacteria in the rhizosphere and bulk soils of control group (C), healthy group (H) and diseased group (D) were similar, being Proteobacteria and Acidobacteriota at the phylum level (Fig. S2 b-c), and Candidatus_Udeaeobacter and unclassified_ Vicinamibacterales at the genus level (Fig. 3 c, e). The dominant fungi in the root of the control group (C) and the healthy group (H) were Basidiomycota at the phylum level (Fig. S2 d) and unclassified_Basidiomycota at the genus level (Fig. 3 b). In the diseased group (D), the dominant fungi in the root were Ascomycota at the phylum level (Fig. S2 d), and Verticillium at the genus level (Fig. 3 b). The dominant bacteria in rhizosphere and bulk soils of control group (C), healthy group (H) and diseased group (D) were similar, being Ascomycota and Basidiomycota at the phylum level (Fig. S2 e-f) and unclassified_fungi at the genus level (Fig. 3 d, f). ------------------------------------------- Fig. 3 ------------------------------------------- Differences in bacterial composition between the control group (C), the healthy group (H) and the diseased group (D) were analyzed using LEfSe (LDA Effect Size). Almost no significant differences were observed between the control group (C) and the healthy group (H) (Fig. 4 a-c). However, compared to the diseased group (D), both the control group (C) and the healthy group (H) were found to be significantly enriched in Bacilli and Clostridia in the roots (Fig. 4 d, g), significantly enriched in Burkholderiales in the rhizosphere soil (Fig. 4 e, h), and significantly enriched in Verrucomicrobiota while Beijerinckiaceae and Rhizobiales were suppressed (Fig. 4 f, i) in the bulk soil. These results suggest that Bacilli , Clostridia , Burkholderiales and Verrucomicrobiota may be potential biocontrol bacteria. ------------------------------------------- Fig. 4 ------------------------------------------- Differences in fungal composition between the control group (C), the healthy group (H) and the diseased group (D) were also analyzed using LEfSe (LDA Effect Size). Few differences were observed between the control group (C) and the healthy group (H) (Fig. 5 a-c). Compared to the diseased group (D), both the control group (C) and the healthy group (H) significantly suppressed Verticillium and Fusarium , while enriched taxa of Archacosporales , Glomerales , unclassified Basidiomycota and unclassified Glomeromycota in the roots (Fig. 5 d, g), both significantly suppressed Entolomataceae in the rhizosphere soil (Fig. 5 e, h), both significantly enriched taxa of Archaeosporales (Fig. 5 f, i) in the bulk soil. These results suggest that the two pathogens ( Verticillium and Fusarium ) may collaborate to induce Verticillium wilt and then black rot, resulting in the shedding of cotton leaves. Furthermore, Archacosporales , Glomerales , unclassified Basidiomycota and unclassified Glomeromycota may serve as potential biocontrol fungi. ------------------------------------------- Fig. 5 ------------------------------------------- Most of the biocontrol strains were Bacillus A total of 34 strains were isolated from the root, including 12 strains that antagonized V. dahliae V991 and 12 strains that antagonized F. oxysporum F679 (Table 4 ). Most of these biocontrol strains were from the control group (C). We isolated 36 strains from the rhizosphere soil, including 5 strains that antagonized V. dahliae V991 and 3 strains that antagonized F. oxysporum F679. 44 strains were isolated from bulk soil, including 3 strains that antagonized V. dahliae V991 and 1 strain that antagonized F. oxysporum F679. Overall, 20 strains inhibited the growth of V. dahliae V991, while 16 strains inhibited the growth of F. oxysporum F679 (Fig. 6 a, c). Table 4 Number and proportion of biocontrol strains in different groups from root, rhizosphere and bulk soil. Strains Biocontrol strains Against V. dahliae V991 Against F. oxysporum F679 Groups Number Number Ratio Number Ratio Root (E) C 15 7 46.67% 7 46.67% H 12 2 16.67% 3 25.00% D 7 3 42.86% 2 28.57% Total 34 12 35.29% 12 35.29% Rhizosphere soil (R) C 10 1 10.00% 2 20.00% H 12 1 8.33% 0 0.00% D 14 3 21.43% 1 7.14% Total 36 5 13.89% 3 8.33% Bulk soil (S) C 12 3 25.00% 1 8.33% H 13 0 0.00% 0 0.00% D 19 0 0.00% 0 0.00% Total 44 3 6.82% 1 2.27 C, control group; H, healthy group; D, diseased group. The strains that antagonistic to V. dahliae V991 and F. oxysporum F679 were 85.00% and 86.67% Bacillus sp., respectively. Most of these biocontrol strains were B. amyloliquefaciens and B. velezensis (Fig. 6 b, d). This suggests that Bacillus could be a good source for selecting biocontrol bacteria. Strain M9 (identified as Bacillus amyloliquefaciens , NCBI accession no. PV449940) exhibited the strongest antagonistic activity against V. dahliae V991 and F. oxysporum F679. Strain R19 (identified as Bacillus cereus , China General Microbiology Culture Collection Center (CGMCC) accession no.27792, NCBI accession no. PV449942) exhibited relatively weaker antagonistic activity against V. dahliae V991. ------------------------------------------- Table 4 ------------------------------------------- ------------------------------------------- Fig. 6 ------------------------------------------- Bacillus cereus R19 effectively controlled cotton Verticillium wilt The strongest biocontrol bacteria ( B. amyloliquefaciens M9, BM) and the weaker ( B. cereus R19, BR) against V. dahliae V991 were selected for pot experiments to test whether the in vivo and in vitro effects were consistent. We also tested 3 different concentrations (10 7 CFU/mL, 10 8 CFU/mL and 10 9 CFU/mL) of biocontrol bacteria to determine whether increasing the concentration of biocontrol bacteria improves their ability to control the disease. The results showed that all treatments except BR8 reduced the disease incidence rate compared to the CK (Fig. 7 a, b). BR9 had the lowest disease incidence rate. BR9 significantly reduced the disease incidence rate by 44.82% compared to CK. BM9, BM7, BR7 and BM8 reduced the disease incidence rate by 24.14%, 20.69%, 13.80% and 6.90% respectively compared to CK. These treatments had no significant effect on cotton dry weight, root length and plant height (Fig. 7 c-e). However, BM9, BM8, BR7, BR9, BM7 and BR8 increased cotton dry weight by 18.46%, 13.40%, 10.74%, 8.33%, 3.47% and 2.24% respectively compared to CK (Fig. 7 c). BM9 and BR9 increased cotton root length by 9.01% and 6.18% respectively compared to CK (Fig. 7 d). BM9, BR7 and BR9 increased cotton root length by 2.16%, 0.92% and 0.87% respectively compared to CK (Fig. 7 e). These results suggest that BM, which has a strong biocontrol function, also exhibits probiotic properties, indicating that the in vivo and in vitro behavior of the biocontrol bacteria is inconsistent and that different concentrations of biocontrol bacteria can affect their biocontrol efficacy. ------------------------------------------- Fig. 7 ------------------------------------------- We conducted cotton field experiments with BR and BM. The results showed that the incidence rate of cotton Verticillium wilt in cotton was significantly lower in the BR than in the CK and BM (Fig. 8 ). BR reduced the disease incidence by 88.46% while BM reduced it by 50.01% compared to CK. ------------------------------------------- Fig. 8 ------------------------------------------- Discussion Few studies have examined the use of comparative microbiomes to guide the screening of biocontrol bacteria for cotton Verticillium wilt. Zeng et al. ( 2022 ) was the first to discover, by comparing the endophytic microbiomes of healthy and diseased (Verticillium wilt) cotton, that Pseudomonas could effectively control cotton Verticillium wilt in pot experiments. In this study, we conducted a comprehensive comparative analysis of the microbiomes in the roots, rhizosphere and bulk soil of healthy and diseased cotton, and found that the number of differentially abundant species in the roots was higher than that in the rhizosphere and bulk soil. Furthermore, a greater number of biocontrol bacteria were identified in the roots than in the rhizosphere and bulk soil. Bacillus amyloliquefaciens M9 (BM), which exhibits the highest antifungal activity, was also screened from roots. This suggests that the roots are a better site for biocontrol bacteria screening than the rhizosphere and bulk soil. The ability of bacterial endophytes to colonise an ecological niche similar to that of vascular wilt pathogens makes them promising candidates for biocontrol of wilt diseases (Eljounaidi et al., 2016 , Song et al., 2024 ). Although numerous studies have screened biocontrol bacteria for cotton Verticillium wilt, only some have conducted pot experiments and even fewer have performed field experiments (Su et al., 2021 ). The biocontrol efficacy of Bacillus generally outperforms that of other species (Yuan et al., 2017 ). Pot experiments have shown that Bacillus , particularly Bacillus subtilis , typically achieves a biocontrol efficiency of 40–90% against cotton Verticillium wilt, while field experiments have indicated that its efficacy generally ranges from 40–70% (Bai et al., 2022 , Zhao et al., 2022 , Song et al., 2024 ). While some studies have shown B. amyloliquefaciens and B. cereus to be effective (~ 80%) in controlling cotton Verticillium wilt in pot experiments, reports on field experiments are lacking (Zhou et al., 2017 , Liu et al., 2023 ). In this study, we screened B. cereus R19 (BR) and B. amyloliquefaciens M9 (BM), which showed excellent biocontrol efficiency against cotton Verticillium wilt in both pot (44.44% and 33.33%, respectively) and field experiments (88.46% and 50.01%, respectively). This suggests that using comparative microbiome analysis to guide the screening of biocontrol bacteria is an effective strategy. However, Bacillus cereus R19 (BR),a strain with weaker antifungal activity that was screened from the rhizosphere, performed better than Bacillus amyloliquefaciens M9 (BM), a strain with the strongest antifungal activity that was screened from roots, in both pot and field experiments. This may be due to R19’s strong adaptability to cotton, soil, and the indigenous microbiome (Deketelaere et al., 2017 , Kaminsky et al., 2019 , Thakur et al., 2024 ). The colonization and function of exogenous inoculants are affected by various soil biotic and abiotic factors, and the specific reasons require further in-depth analysis. The comparative microbiome analysis indicates that Bacilli , Clostridia , Archacosporales , Glomerales , unclassified Basidiomycota , unclassified Glomeromycota , Burkholderiales and Verrucomicrobiota are potential biocontrol bacteria. These have been supported by several studies (Zhang et al., 2022 , Qi et al., 2024 , Wu et al., 2024 ). Many studies have demonstrated that species in the Bacilli , such as Bacillus subtilis and Bacillus amyloliquefaciens , have the ability to control cotton Verticillium wilt (Zhao et al., 2022 , Liu et al., 2023 ). Members of Glomerales , such as Rhizophagus and Glomus , have also been reported to alleviate cotton Verticillium wilt (Kobra et al., 2011 , Orak & Demir, 2011 , Zhang et al., 2018 ). While there are no reports of Clostridia , unclassified Basidiomycota or Burkholderiales controlling cotton Verticillium wilt, these groups have been shown to control other plant diseases. For example, some studies have shown that Clostridium beijerinckii in Clostridia can control Fusarium wilt and root-knot nematodes (Ueki et al., 2017 , Lian et al., 2022 ). Certain Basidiomycota species can control take-all disease in wheat (Gholami et al., 2019 , Sandargo et al., 2019 ). Burkholderia , which are common biocontrol agents In Burkholderiales , can control plant diseases such as rice blast and black rot (Pal et al., 2022 , Xue et al., 2022 , Yu et al., 2022 ). There are no reports on the biocontrol effects of Archaeosporales or Verrucomicrobiota , but Archaeosporales is an order of arbuscular mycorrhiza fungi (AMF) (Kruger et al., 2012 ) and Verrucomicrobiota is one of the main groups of soil prokaryotes (Rakitin et al., 2024 ). Both of these groups may play important roles in plant health. The biocontrol bacteria that we screened were mainly Bacillus , with a small amount of Pseudomonas . This may be due to the difficulty in culturing other bacteria. In future studies, culture methods could be optimized to obtain these potential biocontrol bacteria (Lewis et al., 2021 , Dai et al., 2025 ). Statements & Declarations Funding This work was supported by the Tianjin Municipal Science and Technology Program (23YFYSHZ00050 and 24PTLYHZ00300), the Central Guidance Fund for Local Scientific and Technological Development (Establishment and key technologies of agricultural microbial resource library of Xinjiang, 2024YD009), the Xinjiang Agricultural Microorganisms and Fertilizer Creation Key Laboratory Open Project (SYS2024-01) and the Tianjin Synthetic Biotechnology Innovation Capacity Improvement Project (TSBICIP-IJCP-001). Competing Interests The authors have no relevant financial or non-financial interests to disclose. Author Contributions Jingjing Wang, Zhiyong Huang, Shiwu Xu, Bo Gao and Wanyi Zhang contributed to the study conception and design. Material preparation, data collection and analysis were performed by Wanyi Zhang, Jingjing Wang Peng Wu, Yan Pang, Shijia Dai, Xiaoxia Zhang and Ying Chen. The first draft of the manuscript was written by Wanyi Zhang and Jingjing Wang and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. Data availability The datasets generated during the current study are available in the National Center for Biotechnology Information (NCBI) repository and supplementary materials. Ethical approval This article does not contain any studies with human participants or animals performed by any of the authors. References Bai H, Feng Z, Zhao L, Feng H, Wei F, Zhou J, Gu A, Zhu H, Peng J & Zhang Y (2022) Efficacy evaluation and mechanism of Bacillus subtilis EBS03 against cotton Verticillium wilt. Journal of Cotton Research 5 : 26. Berg G (2009) Plant-microbe interactions promoting plant growth and health: perspectives for controlled use of microorganisms in agriculture. Applied Microbiology and Biotechnology 84 : 11-18. 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Zeng Q, Man X, Dai Y & Liu H (2022) Pseudomonas spp. Enriched in endophytic community of healthy cotton plants inhibit cotton Verticillium wilt. Frontiers in Microbiology 13 : 906732. Zhang Q, Gao X, Ren Y, Ding X, Qiu J, Li N, Zeng F & Chu Z (2018) Improvement of Verticillium wilt resistance by applying arbuscular mycorrhizal fungi to a cotton variety with high symbiotic efficiency under field conditions. International Journal of Molecular Sciences 19 : 241. Zhang Y, Zhou J, Zhao L, Feng Z, Wei F, Bai H, Feng H & Zhu H (2022) A review of the pathogenicity mechanism of Verticillium dahliae in cotton. Journal of Cotton Research 5 : 3. Zhang Y, Yang Y, Yu L, Wang A, Xue C, Zhang J, Duan A & Zhao M (2022) Composition and characteristics of soil microbial communities in cotton fields with different incidences of Verticillium wilt. Plant signaling & behavior 17 : 2034271. Zhang Y, Xue C, Wang X, Zhang J, Wang A, Yang Y, Jia X, Zhang W & Zhao M (2024) The control effect of endophytic fungus Fusarium equiseti FUS-8 on cotton Verticillium wilt and its effects on soil microbial communities. Plant and Soil . Zhang Y, Zhao L, Li D, Li Z, Feng H, Feng Z, Wei F, Zhou J, Ma Z, Yang J & Zhu H (2025) A comprehensive review on elucidating the host disease resistance mechanism from the perspective of the interaction between cotton and Verticillium dahliae . Journal of Cotton Research 8 : 5. Zhao W, Guo Q, Li S, Lu X, Dong L, Wang P, Zhang X, Su Z & Ma P (2022) Application of Bacillus subtilis NCD-2 can suppress cotton verticillium wilt and its effect on abundant and rare microbial communities in rhizosphere. Biological Control 165 : 104812. Zhou J, Feng Z, Liu S, Wei F, Shi Y, Zhao L, Huang W, Zhou Y, Feng H & Zhu H (2021) CGTase, a novel antimicrobial protein from Bacillus cereus YUPP-10, suppresses Verticillium dahliae and mediates plant defence responses. Molecular Plant Pathology 22 : 130-144. Zhou JF, ZiLi, Feng H, Li Y, Yuan Y, Li Z, Wei F, Shi Y, Zhao L, Sun Z, Zhu H & Zhou Y (2017) Biocontrol effect and mechanism of cotton endophytic bacterium Bacillus cereus YUPP-10 against Verticillium wilt in Gossypium hirsutum . Scientia Agricultura Sinica 50 : 2717-2727. Zhu Y, Zhao M, Li T, Wang L, Liao C, Liu D, Zhang H, Zhao Y, Liu L, Ge X & Li B (2023) Interactions between Verticillium dahliae and cotton: pathogenic mechanism and cotton resistance mechanism to Verticillium wilt. Frontiers in Plant Science 14 : 1174281. Additional Declarations No competing interests reported. Supplementary Files 20250613Supplymaterials.docx 20250613RawdataFig1678.xlsx 20250613RawdataFig2shannonindex.xlsx 20250613RawdataFig3relabundancetable.xlsx 20250613RawdataFig2PCoA.xlsx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board 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-6884856","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":472686451,"identity":"968926f5-3ed3-43bc-84c9-1cec2115d391","order_by":0,"name":"Wanyi Zhang","email":"","orcid":"","institution":"Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Wanyi","middleName":"","lastName":"Zhang","suffix":""},{"id":472686452,"identity":"ac682062-4dcf-439e-b708-61cb3e806f6e","order_by":1,"name":"Jingjing 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1","display":"","copyAsset":false,"role":"figure","size":16622932,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eV. dahliae \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eV991 inoculation on cotton seedlings. \u003c/strong\u003eGrowth of cotton seedlings in pot experiments (a) and the disease incidence rate (b) and disease index (c) in different treatments. CK, non-inoculation; V991, \u003cem\u003eV. dahliae \u003c/em\u003eV991 inoculation; C, control group; H, healthy group; D, diseased group.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6884856/v1/6abd9b8703ae76700e248e7e.png"},{"id":84884480,"identity":"0c024291-2178-412d-b88b-2f8e2b0466c1","added_by":"auto","created_at":"2025-06-18 11:28:17","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2913009,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe α diversity (a-f) and β diversity (g-l) of bacterial (a-c, g-i) and fungal (d-f, j-l) communities in root (a, d, g, j), rhizosphere soil (b, e, h, k) and bulk soil (c, f, i, l).\u003c/strong\u003eC, control group; H, healthy group; D, diseased group. Asterisks indicate statistically significant (*\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; ***\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6884856/v1/af8cc22687a55640f88319b4.png"},{"id":84884478,"identity":"12dea315-e9cb-49bd-bdd3-2d76f3489ba3","added_by":"auto","created_at":"2025-06-18 11:28:17","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2145220,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe relative abundance of bacteria (a, c, e) and fungi (b, d, f) in root (a, b), rhizosphere soil (c, d) and bulk soil (e, f) at the genus level.\u003c/strong\u003e The top ten genus are shown in the figure, with other genus grouped together under others. C, control group; H, healthy group; D, diseased group.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6884856/v1/910512b5ce669b0ce4e81399.png"},{"id":84883427,"identity":"34b44d6a-751e-4809-a1e3-d32165bf9ff7","added_by":"auto","created_at":"2025-06-18 11:20:17","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":6947781,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDifferential bacteria (LDA \u0026gt; 4) between control and healthy group (a-c), control and diseased group (d-f) and healthy and diseased group (g-i) in root (a, d, g), rhizosphere soil (b, e, h) and bulk soil (e, f, i).\u003c/strong\u003eC, control group; H, healthy group; D, diseased group.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6884856/v1/1cbb9c7693062e8bb3dcc865.png"},{"id":84885101,"identity":"100a66ea-229c-40d2-9af7-51e6858e069e","added_by":"auto","created_at":"2025-06-18 11:36:17","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":8209682,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDifferential fungi (LDA \u0026gt; 4) between control and healthy group (a-c), control and diseased group (d-f) and healthy and diseased group (g-i) in root (a, d, g), rhizosphere soil (b, e, h) and bulk soil (e, f, i).\u003c/strong\u003eC, control group; H, healthy group; D, diseased\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6884856/v1/a6b1a9b27a4bd181f9169200.png"},{"id":84884482,"identity":"ff7effdb-dff4-47fd-bb1f-5b322f9330c2","added_by":"auto","created_at":"2025-06-18 11:28:17","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2473383,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBiocontrol strains antagonizing \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eVerticillum dahliae \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eV991 (a, b) and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eFusarium oxysporum\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e F679 (c, d). \u003c/strong\u003eThe diameter of the biocontrol inhibition zone against \u003cem\u003eV. dahliae \u003c/em\u003eV991 (a) and \u003cem\u003eF. oxysporum\u003c/em\u003e F679 (c); the species of the biocontrol strains inhibiting \u003cem\u003eV. dahliae \u003c/em\u003eV991 (b) and \u003cem\u003eF. oxysporum\u003c/em\u003e F679 (d). T, strains from bulk soil; R, strains from rhizosphere soil; M, strains from root.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6884856/v1/990dc8ca07aafe3e6ab73a7e.png"},{"id":84884489,"identity":"d99ea226-c36b-4d5d-b7a7-316e986e35de","added_by":"auto","created_at":"2025-06-18 11:28:18","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":28484431,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of different biocontrol bacteria on cotton phenotype(a), disease incidence rate (b), dry weight (c), root length (d) and plant height (e).\u003c/strong\u003e CK, germinating seeds soaked in sterile water; BM7, BM8, BM9, germinating seeds soaked in \u003cem\u003eB. amyloliquefaciens\u003c/em\u003e M9 at 10\u003csup\u003e7\u003c/sup\u003e CFU/mL, 10\u003csup\u003e8\u003c/sup\u003e CFU/mL, 10\u003csup\u003e9\u003c/sup\u003e CFU/mL concentrations respectively; BR7, BR8, BR9, germinating seeds soaked in \u003cem\u003eB. cereus\u003c/em\u003e R19 at 10\u003csup\u003e7\u003c/sup\u003e CFU/mL, 10\u003csup\u003e8\u003c/sup\u003e CFU/mL, 10\u003csup\u003e9\u003c/sup\u003e CFU/mL concentrations respectively. Different letters indicate significant differences between treatments (\u003cem\u003eP\u003c/em\u003e ≤ 0.05).\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-6884856/v1/22b824ace9abab6a441f9853.png"},{"id":84883439,"identity":"69c0ff48-d943-465d-8933-eb784f0bb639","added_by":"auto","created_at":"2025-06-18 11:20:18","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":11128172,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of different biocontrol bacteria on cotton Verticillium wilt in the field.\u003c/strong\u003e a, cotton Verticillium wilt disease incidence rate; b, diseased plant; c, healthy plant. CK, non-inoculation; BR, inoculation with \u003cem\u003eB. cereus\u003c/em\u003e R19; BM, inoculation with \u003cem\u003eB. amyloliquefaciens\u003c/em\u003e M9. Different letters indicate significant differences between treatments (\u003cem\u003eP\u003c/em\u003e≤ 0.05).\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-6884856/v1/58d00ce10eaf6bb1fba904ed.png"},{"id":88468963,"identity":"ef013a6b-b2d4-4dd0-8ce9-02b99246b534","added_by":"auto","created_at":"2025-08-06 18:17:20","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":75963190,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6884856/v1/c12d833f-e11a-4726-b6b1-5b4d0e08c2ad.pdf"},{"id":84883420,"identity":"7b62dd88-0f90-4efd-b983-d04c4f045119","added_by":"auto","created_at":"2025-06-18 11:20:17","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":417531,"visible":true,"origin":"","legend":"","description":"","filename":"20250613Supplymaterials.docx","url":"https://assets-eu.researchsquare.com/files/rs-6884856/v1/1f3dcc3e3e8e961c84cd7b67.docx"},{"id":84883417,"identity":"a4d6fe1b-61bc-4af3-b2da-97698f19654f","added_by":"auto","created_at":"2025-06-18 11:20:17","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":16881,"visible":true,"origin":"","legend":"","description":"","filename":"20250613RawdataFig1678.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6884856/v1/d8e290e4dd0c53ee6627941c.xlsx"},{"id":84883424,"identity":"b2a1fb9b-59cb-4b2c-b959-32458798377c","added_by":"auto","created_at":"2025-06-18 11:20:17","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":16942,"visible":true,"origin":"","legend":"","description":"","filename":"20250613RawdataFig2shannonindex.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6884856/v1/bdc7c2818434271928dd7a03.xlsx"},{"id":84884481,"identity":"b554bbdb-3ca0-4b11-814c-3ed7a41d8b47","added_by":"auto","created_at":"2025-06-18 11:28:17","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":18879,"visible":true,"origin":"","legend":"","description":"","filename":"20250613RawdataFig3relabundancetable.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6884856/v1/4ccbd4f62335002b223885f3.xlsx"},{"id":84883436,"identity":"fc84ba8e-80bd-4edd-b427-1c0189fe579c","added_by":"auto","created_at":"2025-06-18 11:20:17","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":21775,"visible":true,"origin":"","legend":"","description":"","filename":"20250613RawdataFig2PCoA.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6884856/v1/6222228d6b1d37150a48c71c.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Development of biocontrol agents for cotton verticillium wilt using microbiome analysis","fulltext":[{"header":"Introduction","content":"\u003cp\u003eChina is the world\u0026rsquo;s largest consumer and second-largest producer of cotton (Feng et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The quality and yield of cotton are severely affected by Verticillium wilt, a soil-borne disease caused by the \u003cem\u003eVerticillium dahliae\u003c/em\u003e pathogen (Zhu et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2023\u003c/span\u003e, Yang et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Unfortunately, Verticillium wilt is currently considered the \u0026lsquo;cancer of cotton\u0026rsquo;. It generally causes plant dysplasia, wilting and yellowing of leaves, and browning of vascular bundles, and eventually leading to early death (Zhang et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In recent years, the incidence of Verticillium wilt has increased, resulting in significant economic losses in the cotton industry (Zhang et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Controlling the wide spread of \u003cem\u003eV. dahliae\u003c/em\u003e remains huge challenge due to its stable dormant microsclerotia structure, long-term variability and coevolution with host plants (Kong et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Although soil fumigation, chemical pesticides and crop rotation are common methods of controlling Verticillium wilt, they cause significant environmental damage and only alleviate diseases to a limited extent, making them unsuitable for large-scale application (Carroll et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Biological control, which leverages antagonistic microorganisms to suppress pathogens, offers a sustainable alternative to chemical interventions (Berg, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). This approach aligns with global efforts to reduce pesticide use and promote eco-friendly agriculture. In the case of cotton Verticillium wilt, potential biocontrol agents include bacteria (e.g., \u003cem\u003eBacillus\u003c/em\u003e spp.) (Zhou et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2021\u003c/span\u003e, Song et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) and non-pathogenic isolates of \u003cem\u003eFusarium\u003c/em\u003e (Zhang et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). These organisms employ various mechanisms to inhibit \u003cem\u003eV. dahliae\u003c/em\u003e, such as competing for resources and producing antifungal metabolites (e.g., lipopeptides, siderophores), as well as inducing systemic resistance in plants. However, effective biocontrol strains for field application are still lacking.\u003c/p\u003e \u003cp\u003eAdvances in high-throughput sequencing and microbiome analysis have revolutionized our understanding of plant-microbe interactions. The soil and root microbiome plays a pivotal role in plant health and agricultural sustainability. These microorganisms form intricate symbiotic relationships with plants, contributing to nutrient acquisition, stress resilience, and pathogen suppression (Trivedi et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Comparative microbiome analysis, a powerful tool for identifying microbial taxa associated with disease resistance, has become an increasingly popular method for guiding the selection of biocontrol strains. Mendes et al. (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2011\u003c/span\u003e) developed a framework for using comparative microbiome analysis to prioritize candidate biocontrol agents. They compared rhizosphere microbiomes from disease-suppressive and conducive soils and identified γ-\u003cem\u003eProteobacteria\u003c/em\u003e, especially \u003cem\u003ePseudomonadaceae\u003c/em\u003e, as key drivers of suppression to \u003cem\u003eRhizoctonia solani\u003c/em\u003e. They then isolated \u003cem\u003ePseudomonas\u003c/em\u003e from suppressive soils and found that it could suppress \u003cem\u003eRhizoctonia solani\u003c/em\u003e in vitro and in greenhouse trials. Kwak et al. (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) conducted comparative analyses of rhizosphere microbiomes from resistant and susceptible tomato enabled the identification and assembly of a flavobacterial genome that was far more abundant in the resistant plant rhizosphere microbiome than in that of the susceptible plant. They cultivated this flavobacterium, named TRM1, and found that it could suppress \u003cem\u003eRalstonia solanacearum\u003c/em\u003e disease development in a susceptible plant in pot experiments. Zeng et al. (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) investigated the endophytic microbiome of healthy and Verticillium wilt infected cotton plants, founding that \u003cem\u003ePseudomonas\u003c/em\u003e was significantly depleted in the diseased plants. They then isolated \u003cem\u003ePseudomonas\u003c/em\u003e from the healthy cotton plants and demonstrated that plants inoculated with \u003cem\u003ePseudomonas\u003c/em\u003e sp. strains showed increased resistance to the cotton Verticillium wilt compared with the control plants in pot experiments. However, there is still a lack of comparative microbiome analysis across multiple sites.\u003c/p\u003e \u003cp\u003eIn this paper, we first investigated whether \u003cem\u003eVerticillium dahliae\u003c/em\u003e V991 causes cotton Verticillium wilt using pot experiments. We then analyzed the microbial communities in the roots, rhizosphere and bulk soil of healthy and diseased cotton plants using amplicon sequencing. Based on these results, we targeted screening biocontrol strains against \u003cem\u003eV. dahliae\u003c/em\u003e 991and tested the ability of typical strains to control Verticillium wilt in pot and field experiments.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003eStrains\u003c/h2\u003e\n \u003cp\u003eThe \u003cem\u003eVerticillium dahliae\u003c/em\u003e V991 strain (National Center for Biotechnology Information (NCBI) accession number PV450007) and \u003cem\u003eFusarium oxysporum\u003c/em\u003e F679 strain (NCBI accession number PV450006) were stored at the Applied Microbial Ecology Engineering Lab at Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003ePathogenic experiment of\u003c/strong\u003e \u003cstrong\u003eV. dahliae\u003c/strong\u003e \u003cstrong\u003eV991\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eThe pathogenicity of \u003cem\u003eV. dahliae\u003c/em\u003e V991 was tested using a cotton pot experiment. The cotton seeds were sterilized with 3% sodium hypochlorite solution for 15 min. The surface-sterilized seeds were germinated in water for 48 h before being sown in nursery substrate (peaty soil: forest humus: vermiculite: perlite (\u003cem\u003ev\u003c/em\u003e:\u003cem\u003ev\u003c/em\u003e)\u0026thinsp;=\u0026thinsp;2:1:1:1) at the cotyledon stage (day 5\u0026ndash;7).\u003c/p\u003e\n \u003cp\u003e\u003cem\u003eV. dahliae\u003c/em\u003e V991 was cultured in PDA medium (Beijing Solarbio Science \u0026amp; Technology Co., Ltd.) at 30℃ for 5 days. The concentration of fungal spores was adjusted to 10\u003csup\u003e5\u003c/sup\u003e CFU/mL using sterile water. On day 7, the 1 cm root tips of cotton seedlings were cut off and soaked in the \u003cem\u003eV. dahliae\u003c/em\u003e V991 spore solution (V991) or sterile water (CK) for 3 min. Each treatment consisted of 30 cotton plants. The pots were then placed randomly in a greenhouse with a relative humidity of 70\u0026ndash;80% and 16 h of light. Before seedling emergence, the temperature was maintained at 30℃. Thereafter, the temperature was maintained at 28℃ for all subsequent stages. The pots were watered with 500 mL every 48 h and their position was changed randomly.\u003c/p\u003e\n \u003cp\u003eThe disease index and incidence rate were recorded in cotton plants on the 25th day after infection by \u003cem\u003eV. dahliae\u003c/em\u003e V991. Disease indices were recorded according to the following criteria: Grade 0, healthy strain with no obvious pathological symptoms. Grade 1, 1\u0026ndash;2 cotyledons show pathological symptoms. Grade 2, 1\u0026ndash;2 true leaves show obvious pathological symptoms. Grade 3, 3 or more true leaves show pathological symptoms or only the heart leaf fall. Grade 4, all leaves fall, the growing point dies or the whole plant dies \u003csup\u003e[30]\u003c/sup\u003e.\u003c/p\u003e\n \u003cdiv id=\"Equa\" class=\"Equation\"\u003e\n \u003cdiv class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\u003cimg 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\"\u003e\u003cbr\u003e\u003c/div\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Equb\" class=\"Equation\"\u003e\u003cbr\u003e\u003c/div\u003e\n \u003cp\u003e\u003cstrong\u003eMicrobial diversity analysis\u003c/strong\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003cp\u003eTwo healthy and two diseased cotton seedlings from the V991 inoculation (V991) were taken as the healthy group (H) and diseased group (D). Two cotton seedlings from the non-inoculation (CK) were taken as control group (C). On day 25 after infection by V991, bulk soil, rhizosphere soil and roots from these three groups (H, D and C) were collected to analyze the microbial structure (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ea). The cotton seedlings were carefully taken from the nursery substrate. Bulk soil, which was loosely attached to the roots, was collected by shaking. The rhizosphere soil, which was adhering to the roots, was collected by washing with 25 mL sterile water and then centrifuging at 2500 g for 5 min. The roots were sterilized by soaking them in 1% sodium hypochlorite solution for 10 min followed by washing them 3\u0026ndash;4 times with sterile water. The roots were then soaked in 75% ethanol for 5 min, after which they were rinsed extensively with sterile water a further 3\u0026ndash;4 times. The sterilized roots were collected as root samples. There were four samples in each group. All samples were immediately stored at -80\u0026deg;C for further analysis.\u003c/p\u003e\n\u003cp\u003eInformation on the microbial community was obtained by performing DNA extraction and sequencing of the samples at Biomarker Technologies Co., Ltd (Beijing, China). All primer pairs provided by the company for sequencing are listed in Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e. The raw sequence data were deposited in the NCBI Sequence Read Archive under the accession number PRJNA1071027. The raw data were processed and analyzed using BMKCloud (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ewww.biocloud.net\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eThe primers used to amplify bacterial and fungal communities\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"4\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eTarget microorganism\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003ePrimer\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSequence\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eEndogenous bacteria\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e335F\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u0026apos;-CADACTCCTACGGGAGGC-3\u0026apos;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e769R\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u0026apos;-ATCCTGTTTGMTMCCCVCRC-3\u0026apos;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\" rowspan=\"2\"\u003e\n \u003cp\u003eEndogenous fungi\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eITS1F\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u0026apos;-CTTGGTCATTTAGAGGAAGTAA-3\u0026apos;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eITS2-Fungal-ad\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u0026apos;-TGCGTTCTTCATCGATGC-3\u0026apos;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eBacteria\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e515F\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u0026apos;-GTGYCAGCMGCCGCGGTAA-3\u0026apos;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e926R\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u0026apos;-CCGYCAATTYMTTTRAGTTT-3\u0026apos;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eFungi\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eITS1F\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u0026apos;-CTTGGTCATTTAGAGGAAGTAA-3\u0026apos;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eITS2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u0026apos;-GCTGCGTTCTTCATCGATGC-3\u0026apos;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003e------------------------------------------- Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e -------------------------------------------\u003c/p\u003e\n\u003ch3\u003eIsolation and identification of bacteria\u003c/h3\u003e\n\u003cp\u003eBacteria were isolated from the roots, rhizosphere soil and bulk soil of cotton plants. Suspensions of the rhizosphere soil and bulk soils were diluted to 10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e and 10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e CFU/mL, respectively. Then 100 \u0026micro;L of each dilution was plated onto Luria-Bertani (LB) medium containing 2.5 \u0026micro;g/mL amphotericin B. The roots were crushed and shaken with 1 mL sterile water. Then 100 \u0026micro;L of the resulting suspensions was plated onto LB containing 2.5 \u0026micro;g/mL amphotericin B. All colonies were picked from the plates and further re-streaked on fresh LB plates for purification. After 48 h of incubation at 30℃, the isolates were selected based on the morphology (colour and size) of the bacterial colonies for 16S rRNA gene sequencing and identification.\u003c/p\u003e\n\u003cp\u003eGenomic DNA was extracted from the bacterial monocultures using a TIANamp Bacteria DNA Kit (TIANGEN Biotech Co., Ltd. Beijing, China) following the manufacturer\u0026rsquo;s instructions. PCR amplification of the 16S rRNA gene was performed using the forward primer 27F (5\u0026apos;-AGAGTTTGATCCTGGCTCAG-3\u0026apos;) and the reverse primer 1492R (5\u0026apos;-TACGACTTAACCCCAATCGC-3\u0026apos;) \u003csup\u003e[31]\u003c/sup\u003e. The PCR cycle consisted of an initial denaturation at 95℃ for 3 min, followed by 34 cycles of denaturation at 95℃ for 30 s, denaturation at 55℃ for 30 s, and extension at 72℃ for 90 s and a final extension at 72℃for 5 min. The PCR products were sequenced by Tsingke Biotechnology Co. Ltd. company (Beijing, China). All bacterial isolates were identified using the BLAST search program and the NCBI database. Highly homologous sequences were aligned and isolates with the matching genes that were more than 97% identical were considered to be the same species. All strains were stored in 25% glycerol solutions and placed in an ultra-low temperature refrigerator at -80℃.\u003c/p\u003e\n\u003ch3\u003eAntifungal activity of the isolated bacteria\u003c/h3\u003e\n\u003cp\u003eThe antagonistic activity of each isolated bacterial strain against \u003cem\u003eV. dahliae\u003c/em\u003e V991 or \u003cem\u003eFusarium oxysporum\u003c/em\u003e F679 (\u003cem\u003eF. oxysporum\u003c/em\u003e F679) was evaluated using the inhibition zone method. A single bacterial colony was inoculated into 1 mL of sterile water to adjust the bacterial concentration to 10\u003csup\u003e9\u003c/sup\u003e CFU/mL. Then, 5 \u0026micro;L of the bacterial solution was added to the PDA medium containing the spread pathogens (10\u003csup\u003e5\u003c/sup\u003e CFU/mL) after 1 h. The plates were sealed with parafilm and incubated for 5 days at 26 ℃. Each treatment included 3 replicates. On day 5, the diameter of the inhibition zone formed by the bacteria growing on the PDA medium with pathogens was measured. The larger the inhibition zone, the stronger the bacteria\u0026rsquo;s antifungal ability.\u003c/p\u003e\n\u003ch3\u003eBiocontrol activity in pot experiments\u003c/h3\u003e\n\u003cp\u003eFor pot experiments, we selected the strongest V991 antagonist, \u003cem\u003eB. amyloliquefaciens\u003c/em\u003e M9 (BM), and the weaker \u003cem\u003eB. cereus\u003c/em\u003e R19 (BR), to verify the difference between \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e antagonism. The bacteria were cultured in LB liquid medium (30℃, 180 rpm) for 48 h, and then diluted to different concentrations (10\u003csup\u003e7\u003c/sup\u003e, 10\u003csup\u003e8\u003c/sup\u003e and 10\u003csup\u003e9\u003c/sup\u003e CFU/mL). The cotton seeds were sterilized with 3% sodium hypochlorite solution for 15 min, after which they were soaked in water for 48 h to accelerate germination. The pot experiments comprised 7 treatments involving germinated cotton seeds that were soaked in \u003cem\u003eB. amyloliquefaciens\u003c/em\u003e M9 (BM) and \u003cem\u003eB. cereus\u003c/em\u003e R19 (BR) at concentrations of 10\u003csup\u003e7\u003c/sup\u003e, 10\u003csup\u003e8\u003c/sup\u003e and 10\u003csup\u003e9\u003c/sup\u003e CFU/mL, respectively (Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e) and in sterile water (CK). Each treatment included 36 seedlings. The germinated cotton seeds of the BM7, BM8 and BM9 were soaked in a \u003cem\u003eB. amyloliquefaciens\u003c/em\u003e M9 (BM) solution at concentrations of 10\u003csup\u003e7\u003c/sup\u003e, 10\u003csup\u003e8\u003c/sup\u003e and 10\u003csup\u003e9\u003c/sup\u003e CFU/mL, respectively. The germinated cotton seeds of the BR7, BR8 and BR9 were soaked in a \u003cem\u003eB. cereus\u003c/em\u003e R19 (BR) solution at concentrations of 10\u003csup\u003e7\u003c/sup\u003e, 10\u003csup\u003e8\u003c/sup\u003e and 10\u003csup\u003e9\u003c/sup\u003e CFU/mL, respectively. All seeds were soaked for 5 h and then sown in the nursery substrate (peaty soil: forest humus: vermiculite: perlite (\u003cem\u003ev\u003c/em\u003e:\u003cem\u003ev\u003c/em\u003e:\u003cem\u003ev\u003c/em\u003e:\u003cem\u003ev\u003c/em\u003e)\u0026thinsp;=\u0026thinsp;2:1:1:1).\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable id=\"Tab2\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eThe treatments in pot experiments\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"3\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eTreatments\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eInoculants\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eConcentration\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCK\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSterile water\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e/\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBM7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eB. amyloliquefaciens\u003c/em\u003e M9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10\u003csup\u003e7\u003c/sup\u003e CFU/mL\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBM8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eB. amyloliquefaciens\u003c/em\u003e M9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10\u003csup\u003e8\u003c/sup\u003e CFU/mL\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBM9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eB. amyloliquefaciens\u003c/em\u003e M9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10\u003csup\u003e9\u003c/sup\u003e CFU/mL\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBR7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eB. cereus\u003c/em\u003e R19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10\u003csup\u003e7\u003c/sup\u003e CFU/mL\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBR8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eB. cereus\u003c/em\u003e R19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10\u003csup\u003e8\u003c/sup\u003e CFU/mL\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBR9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eB. cereus\u003c/em\u003e R19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10\u003csup\u003e9\u003c/sup\u003e CFU/mL\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003e------------------------------------------- Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e -------------------------------------------\u003c/p\u003e\n\u003cp\u003eOn day 10, the cotton seedlings, which had two unfolded cotyledons, were carefully removed from the nursery substrate. The root tips of all the cotton seedlings were cut to 1 cm, and the wounded roots were infected with 100 mL of V991 (10\u003csup\u003e5\u003c/sup\u003e CFU/mL). On day 25, plants from each pot were harvested, carefully separated into roots and shoots after gentle washing with tap water, and growth parameters, including disease incidence rate, root length and plant height and dry weight were determined. The lower disease incidence rate indicated the stronger biocontrol ability of the tested bacteria. All statistical tests performed in this study were considered significant at \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003eBiocontrol activity in field experiments\u003c/h2\u003e\n \u003cp\u003eThe biocontrol activity of \u003cem\u003eB. amyloliquefaciens\u003c/em\u003e M9 (BM) and \u003cem\u003eB. cereus\u003c/em\u003e R19 (BR) was tested in field experiments. The bacteria were cultured in LB liquid medium at 30℃, 180 rpm for 48 h, resulting in a final concentrations of approximately 1\u0026times;10\u003csup\u003e10\u003c/sup\u003e CFU/mL. The field experiments were conducted in Beiquan Town, Shihezi City, Xinjiang, China. Three treatments were carried out, CK (inoculation with water), BM (inoculation with \u003cem\u003eB. amyloliquefaciens\u003c/em\u003e M9) and BR (inoculation with \u003cem\u003eB. cereus\u003c/em\u003e R19). Three plots were established for each treatment. Each plot was 4 m\u003csup\u003e2\u003c/sup\u003e in size. One month after sowing the cotton, 30 mL of microbial inoculant or water was applied to each plot. During the cotton harvest, 10 cotton plants were randomly selected from each plot and the incidence rate of Verticillum wilt was counted using the pole-throwing method for each treatment.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eInoculation with\u003c/strong\u003e \u003cstrong\u003eV. dahliae\u003c/strong\u003e \u003cstrong\u003eV991 caused severe verticillium wilt in cotton\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTen days after inoculation with \u003cem\u003eV. dahliae\u003c/em\u003e V991 (V991), the cotton leaves wilted and the roots and stems showed signs of black rot (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ea). After 25 days, the disease incidence rate in the cotton seedlings reached 90%, with an incidence index of 77.5 (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eb-c). In contrast, all cotton seedlings in the non-inoculation group (CK) remained healthy. These results strongly suggest that \u003cem\u003eV. dahliae\u003c/em\u003e V991 is the causal agent of Verticillium wilt in cotton.\u003c/p\u003e\n\u003cp\u003e------------------------------------------- Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e -------------------------------------------\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInoculation with\u003c/strong\u003e \u003cstrong\u003eV. dahliae\u003c/strong\u003e \u003cstrong\u003eV991 altered microbial diversity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eInterestingly, two of the cotton seedlings in the \u003cem\u003eV. dahliae\u003c/em\u003e V991 inoculation (V991) exhibited resistance to the disease. These two cotton seedlings were collected to form the healthy group (H). Two cotton seedlings from the non-inoculation group (CK) were then collected as the control group (C) and two from the \u003cem\u003eV. dahliae\u003c/em\u003e V991 inoculation (V991) as the disease group (D) (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ea). Bulk soil, rhizosphere soil and roots from these three groups (H, D and C) were collected on day 25 after infection by \u003cem\u003eV. dahliae\u003c/em\u003e V991 to analyze the microbial structure. The results showed that the effect of location on microbial diversity far outweighs the effect of disease severity (Fig. \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e). Bacterial and fungal \u0026alpha; diversity (Shannon index) was lower in the disease group (D) than in the healthy group (H) and the control group (C) (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ea-f). However, only endogenous bacterial \u0026alpha; diversity was significantly lower in the disease group (D) compared to the healthy group (H) and the control group (C) (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ea). Endogenous fungal \u0026alpha; diversity was significantly lower in the control group (C) than in the healthy group (H) (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ed). No significant differences were observed in the \u0026alpha; diversity of bacteria and fungi in the rhizosphere and bulk soil among the three groups (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eb-c and Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ee-f).\u003c/p\u003e\n\u003cp\u003eSignificant differences in the \u0026beta; diversity of the bacterial and fungal communities were observed in the root, rhizosphere soil and bulk soils (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eg-l). The difference in bacterial communities in bulk soil was obviously greater than in the root and rhizosphere soil (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eg-i). The difference in fungal communities from the roots was obviously greater than that from the rhizosphere soil and bulk soils (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ej-l). The differences in bacterial and fungal communities between the diseased and control groups (D-C) and between the diseased and healthy groups (D-H) were greater than those between the healthy and control groups (H-C) (Table \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). These results suggest that infection with \u003cem\u003eV. dahliae\u003c/em\u003e V991 altered the diversity of the fungal and bacterial communities.\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable id=\"Tab3\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003ePermutational multivariate analysis of variance (PERMANOVA) of microbial \u0026beta; diversity\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"6\"\u003e\u003c/colgroup\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\" rowspan=\"2\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eBacteria\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eFungi\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eP\u003c/em\u003e-value\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eP\u003c/em\u003e-value\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003eRoot (E)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC-H\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.149\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.031\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.270\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.098\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC-D\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.157\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.046\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.494\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eH-D\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.159\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.023\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.504\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.027\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003eRhizosphere soil (R)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC-H\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.189\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.087\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.206\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.027\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC-D\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.250\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.030\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.217\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eH-D\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.252\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.032\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.207\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003eBulk soil (S)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC-H\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.157\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.030\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.159\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.038\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC-D\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.249\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.183\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eH-D\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.330\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.190\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.032\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"6\"\u003eC, control group; H, healthy group; D, diseased group.\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003e------------------------------------------- Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e -------------------------------------------\u003c/p\u003e\n\u003cp\u003e------------------------------------------- Table \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e -------------------------------------------\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInoculation with\u003c/strong\u003e \u003cstrong\u003eV. dahliae\u003c/strong\u003e \u003cstrong\u003eV991 altered the microbial composition\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe dominant bacteria in the root of the control group (C) and the healthy group (H) were \u003cem\u003eProteobacteria\u003c/em\u003e and \u003cem\u003eFirmicutes\u003c/em\u003e at the phylum level (Fig. \u003cspan class=\"InternalRef\"\u003eS2\u003c/span\u003ea), and \u003cem\u003euncultured_Mollicutes_bacterium\u003c/em\u003e and \u003cem\u003eCandidatus_Solibacter\u003c/em\u003e at the genus level (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ea). In the diseased group (D), the dominant bacteria in the roots were \u003cem\u003eProteobacteria\u003c/em\u003e and \u003cem\u003eMyxococcota\u003c/em\u003e at the phylum level (Fig. \u003cspan class=\"InternalRef\"\u003eS2\u003c/span\u003ea), and \u003cem\u003eAllorhizobium\u003c/em\u003e_\u003cem\u003eNeorhizobium\u003c/em\u003e_\u003cem\u003ePararhizobium\u003c/em\u003e_\u003cem\u003eRhizobium\u003c/em\u003e and \u003cem\u003ePseudomonas\u003c/em\u003e at the genus level (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ea). The dominant bacteria in the rhizosphere and bulk soils of control group (C), healthy group (H) and diseased group (D) were similar, being \u003cem\u003eProteobacteria\u003c/em\u003e and \u003cem\u003eAcidobacteriota\u003c/em\u003e at the phylum level (Fig. \u003cspan class=\"InternalRef\"\u003eS2\u003c/span\u003eb-c), and \u003cem\u003eCandidatus_Udeaeobacter\u003c/em\u003e and unclassified_\u003cem\u003eVicinamibacterales\u003c/em\u003e at the genus level (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ec, e).\u003c/p\u003e\n\u003cp\u003eThe dominant fungi in the root of the control group (C) and the healthy group (H) were \u003cem\u003eBasidiomycota\u003c/em\u003e at the phylum level (Fig. \u003cspan class=\"InternalRef\"\u003eS2\u003c/span\u003ed) and \u003cem\u003eunclassified_Basidiomycota\u003c/em\u003e at the genus level (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eb). In the diseased group (D), the dominant fungi in the root were \u003cem\u003eAscomycota\u003c/em\u003e at the phylum level (Fig. \u003cspan class=\"InternalRef\"\u003eS2\u003c/span\u003ed), and \u003cem\u003eVerticillium\u003c/em\u003e at the genus level (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eb). The dominant bacteria in rhizosphere and bulk soils of control group (C), healthy group (H) and diseased group (D) were similar, being \u003cem\u003eAscomycota\u003c/em\u003e and \u003cem\u003eBasidiomycota\u003c/em\u003e at the phylum level (Fig. \u003cspan class=\"InternalRef\"\u003eS2\u003c/span\u003ee-f) and \u003cem\u003eunclassified_fungi\u003c/em\u003e at the genus level (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ed, f).\u003c/p\u003e\n\u003cp\u003e------------------------------------------- Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e -------------------------------------------\u003c/p\u003e\n\u003cp\u003eDifferences in bacterial composition between the control group (C), the healthy group (H) and the diseased group (D) were analyzed using LEfSe (LDA Effect Size). Almost no significant differences were observed between the control group (C) and the healthy group (H) (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ea-c). However, compared to the diseased group (D), both the control group (C) and the healthy group (H) were found to be significantly enriched in \u003cem\u003eBacilli\u003c/em\u003e and \u003cem\u003eClostridia\u003c/em\u003e in the roots (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ed, g), significantly enriched in \u003cem\u003eBurkholderiales\u003c/em\u003e in the rhizosphere soil (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ee, h), and significantly enriched in \u003cem\u003eVerrucomicrobiota\u003c/em\u003e while \u003cem\u003eBeijerinckiaceae\u003c/em\u003e and \u003cem\u003eRhizobiales\u003c/em\u003e were suppressed (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ef, i) in the bulk soil. These results suggest that \u003cem\u003eBacilli\u003c/em\u003e, \u003cem\u003eClostridia\u003c/em\u003e, \u003cem\u003eBurkholderiales\u003c/em\u003e and \u003cem\u003eVerrucomicrobiota\u003c/em\u003e may be potential biocontrol bacteria.\u003c/p\u003e\n\u003cp\u003e------------------------------------------- Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e -------------------------------------------\u003c/p\u003e\n\u003cp\u003eDifferences in fungal composition between the control group (C), the healthy group (H) and the diseased group (D) were also analyzed using LEfSe (LDA Effect Size). Few differences were observed between the control group (C) and the healthy group (H) (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ea-c). Compared to the diseased group (D), both the control group (C) and the healthy group (H) significantly suppressed \u003cem\u003eVerticillium\u003c/em\u003e and \u003cem\u003eFusarium\u003c/em\u003e, while enriched taxa of \u003cem\u003eArchacosporales\u003c/em\u003e, \u003cem\u003eGlomerales\u003c/em\u003e, \u003cem\u003eunclassified Basidiomycota\u003c/em\u003e and \u003cem\u003eunclassified Glomeromycota\u003c/em\u003e in the roots (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ed, g), both significantly suppressed \u003cem\u003eEntolomataceae\u003c/em\u003e in the rhizosphere soil (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ee, h), both significantly enriched taxa of \u003cem\u003eArchaeosporales\u003c/em\u003e (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ef, i) in the bulk soil. These results suggest that the two pathogens (\u003cem\u003eVerticillium\u003c/em\u003e and \u003cem\u003eFusarium\u003c/em\u003e) may collaborate to induce Verticillium wilt and then black rot, resulting in the shedding of cotton leaves. Furthermore, \u003cem\u003eArchacosporales\u003c/em\u003e, \u003cem\u003eGlomerales\u003c/em\u003e, \u003cem\u003eunclassified Basidiomycota\u003c/em\u003e and \u003cem\u003eunclassified Glomeromycota\u003c/em\u003e may serve as potential biocontrol fungi.\u003c/p\u003e\n\u003cp\u003e------------------------------------------- Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e -------------------------------------------\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMost of the biocontrol strains were\u003c/strong\u003e \u003cstrong\u003eBacillus\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA total of 34 strains were isolated from the root, including 12 strains that antagonized \u003cem\u003eV. dahliae\u003c/em\u003e V991 and 12 strains that antagonized \u003cem\u003eF. oxysporum\u003c/em\u003e F679 (Table \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e). Most of these biocontrol strains were from the control group (C). We isolated 36 strains from the rhizosphere soil, including 5 strains that antagonized \u003cem\u003eV. dahliae\u003c/em\u003e V991 and 3 strains that antagonized \u003cem\u003eF. oxysporum\u003c/em\u003e F679. 44 strains were isolated from bulk soil, including 3 strains that antagonized \u003cem\u003eV. dahliae\u003c/em\u003e V991 and 1 strain that antagonized \u003cem\u003eF. oxysporum\u003c/em\u003e F679. Overall, 20 strains inhibited the growth of \u003cem\u003eV. dahliae\u003c/em\u003e V991, while 16 strains inhibited the growth of \u003cem\u003eF. oxysporum\u003c/em\u003e F679 (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ea, c).\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable id=\"Tab4\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eNumber and proportion of biocontrol strains in different groups from root, rhizosphere and bulk soil.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"7\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eStrains\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"4\"\u003e\n \u003cp\u003eBiocontrol strains\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eAgainst \u003cem\u003eV. dahliae\u003c/em\u003e V991\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eAgainst \u003cem\u003eF. oxysporum\u003c/em\u003e F679\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eGroups\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eNumber\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eNumber\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eRatio\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eNumber\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eRatio\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003eRoot (E)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e46.67%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e46.67%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e16.67%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e25.00%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e42.86%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e28.57%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTotal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e35.29%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e35.29%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003eRhizosphere soil (R)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10.00%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e20.00%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8.33%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.00%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e21.43%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.14%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTotal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e36\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e13.89%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8.33%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003eBulk soil (S)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e25.00%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8.33%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.00%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.00%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.00%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.00%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTotal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e44\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.82%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.27\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"7\"\u003eC, control group; H, healthy group; D, diseased group.\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eThe strains that antagonistic to \u003cem\u003eV. dahliae\u003c/em\u003e V991 and \u003cem\u003eF. oxysporum\u003c/em\u003e F679 were 85.00% and 86.67% \u003cem\u003eBacillus\u003c/em\u003e sp., respectively. Most of these biocontrol strains were \u003cem\u003eB. amyloliquefaciens\u003c/em\u003e and \u003cem\u003eB. velezensis\u003c/em\u003e (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eb, d). This suggests that \u003cem\u003eBacillus\u003c/em\u003e could be a good source for selecting biocontrol bacteria. Strain M9 (identified as \u003cem\u003eBacillus amyloliquefaciens\u003c/em\u003e, NCBI accession no. PV449940) exhibited the strongest antagonistic activity against \u003cem\u003eV. dahliae\u003c/em\u003e V991 and \u003cem\u003eF. oxysporum\u003c/em\u003e F679. Strain R19 (identified as \u003cem\u003eBacillus cereus\u003c/em\u003e, China General Microbiology Culture Collection Center (CGMCC) accession no.27792, NCBI accession no. PV449942) exhibited relatively weaker antagonistic activity against \u003cem\u003eV. dahliae\u003c/em\u003e V991.\u003c/p\u003e\n\u003cp\u003e------------------------------------------- Table \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e -------------------------------------------\u003c/p\u003e\n\u003cp\u003e------------------------------------------- Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e -------------------------------------------\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBacillus cereus\u003c/strong\u003e \u003cstrong\u003eR19 effectively controlled cotton Verticillium wilt\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe strongest biocontrol bacteria (\u003cem\u003eB. amyloliquefaciens\u003c/em\u003e M9, BM) and the weaker (\u003cem\u003eB. cereus\u003c/em\u003e R19, BR) against \u003cem\u003eV. dahliae\u003c/em\u003e V991 were selected for pot experiments to test whether the \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e effects were consistent. We also tested 3 different concentrations (10\u003csup\u003e7\u003c/sup\u003e CFU/mL, 10\u003csup\u003e8\u003c/sup\u003e CFU/mL and 10\u003csup\u003e9\u003c/sup\u003e CFU/mL) of biocontrol bacteria to determine whether increasing the concentration of biocontrol bacteria improves their ability to control the disease. The results showed that all treatments except BR8 reduced the disease incidence rate compared to the CK (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003ea, b). BR9 had the lowest disease incidence rate. BR9 significantly reduced the disease incidence rate by 44.82% compared to CK. BM9, BM7, BR7 and BM8 reduced the disease incidence rate by 24.14%, 20.69%, 13.80% and 6.90% respectively compared to CK. These treatments had no significant effect on cotton dry weight, root length and plant height (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003ec-e). However, BM9, BM8, BR7, BR9, BM7 and BR8 increased cotton dry weight by 18.46%, 13.40%, 10.74%, 8.33%, 3.47% and 2.24% respectively compared to CK (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003ec). BM9 and BR9 increased cotton root length by 9.01% and 6.18% respectively compared to CK (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003ed). BM9, BR7 and BR9 increased cotton root length by 2.16%, 0.92% and 0.87% respectively compared to CK (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003ee). These results suggest that BM, which has a strong biocontrol function, also exhibits probiotic properties, indicating that the \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e behavior of the biocontrol bacteria is inconsistent and that different concentrations of biocontrol bacteria can affect their biocontrol efficacy.\u003c/p\u003e\n\u003cp\u003e------------------------------------------- Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e -------------------------------------------\u003c/p\u003e\n\u003cp\u003eWe conducted cotton field experiments with BR and BM. The results showed that the incidence rate of cotton Verticillium wilt in cotton was significantly lower in the BR than in the CK and BM (Fig. \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003e). BR reduced the disease incidence by 88.46% while BM reduced it by 50.01% compared to CK.\u003c/p\u003e\n\u003cp\u003e------------------------------------------- Fig. \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003e -------------------------------------------\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eFew studies have examined the use of comparative microbiomes to guide the screening of biocontrol bacteria for cotton Verticillium wilt. Zeng et al. (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) was the first to discover, by comparing the endophytic microbiomes of healthy and diseased (Verticillium wilt) cotton, that \u003cem\u003ePseudomonas\u003c/em\u003e could effectively control cotton Verticillium wilt in pot experiments. In this study, we conducted a comprehensive comparative analysis of the microbiomes in the roots, rhizosphere and bulk soil of healthy and diseased cotton, and found that the number of differentially abundant species in the roots was higher than that in the rhizosphere and bulk soil. Furthermore, a greater number of biocontrol bacteria were identified in the roots than in the rhizosphere and bulk soil. \u003cem\u003eBacillus amyloliquefaciens\u003c/em\u003e M9 (BM), which exhibits the highest antifungal activity, was also screened from roots. This suggests that the roots are a better site for biocontrol bacteria screening than the rhizosphere and bulk soil. The ability of bacterial endophytes to colonise an ecological niche similar to that of vascular wilt pathogens makes them promising candidates for biocontrol of wilt diseases (Eljounaidi et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2016\u003c/span\u003e, Song et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAlthough numerous studies have screened biocontrol bacteria for cotton Verticillium wilt, only some have conducted pot experiments and even fewer have performed field experiments (Su et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The biocontrol efficacy of \u003cem\u003eBacillus\u003c/em\u003e generally outperforms that of other species (Yuan et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Pot experiments have shown that \u003cem\u003eBacillus\u003c/em\u003e, particularly \u003cem\u003eBacillus subtilis\u003c/em\u003e, typically achieves a biocontrol efficiency of 40\u0026ndash;90% against cotton Verticillium wilt, while field experiments have indicated that its efficacy generally ranges from 40\u0026ndash;70% (Bai et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2022\u003c/span\u003e, Zhao et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2022\u003c/span\u003e, Song et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). While some studies have shown \u003cem\u003eB. amyloliquefaciens\u003c/em\u003e and \u003cem\u003eB. cereus\u003c/em\u003e to be effective (~\u0026thinsp;80%) in controlling cotton Verticillium wilt in pot experiments, reports on field experiments are lacking (Zhou et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2017\u003c/span\u003e, Liu et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In this study, we screened \u003cem\u003eB. cereus\u003c/em\u003e R19 (BR) and \u003cem\u003eB. amyloliquefaciens\u003c/em\u003e M9 (BM), which showed excellent biocontrol efficiency against cotton Verticillium wilt in both pot (44.44% and 33.33%, respectively) and field experiments (88.46% and 50.01%, respectively). This suggests that using comparative microbiome analysis to guide the screening of biocontrol bacteria is an effective strategy. However, \u003cem\u003eBacillus cereus\u003c/em\u003e R19 (BR),a strain with weaker antifungal activity that was screened from the rhizosphere, performed better than \u003cem\u003eBacillus amyloliquefaciens\u003c/em\u003e M9 (BM), a strain with the strongest antifungal activity that was screened from roots, in both pot and field experiments. This may be due to R19\u0026rsquo;s strong adaptability to cotton, soil, and the indigenous microbiome (Deketelaere et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2017\u003c/span\u003e, Kaminsky et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2019\u003c/span\u003e, Thakur et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The colonization and function of exogenous inoculants are affected by various soil biotic and abiotic factors, and the specific reasons require further in-depth analysis.\u003c/p\u003e \u003cp\u003eThe comparative microbiome analysis indicates that \u003cem\u003eBacilli\u003c/em\u003e, \u003cem\u003eClostridia\u003c/em\u003e, \u003cem\u003eArchacosporales\u003c/em\u003e, \u003cem\u003eGlomerales\u003c/em\u003e, \u003cem\u003eunclassified Basidiomycota\u003c/em\u003e, unclassified \u003cem\u003eGlomeromycota\u003c/em\u003e, \u003cem\u003eBurkholderiales\u003c/em\u003e and \u003cem\u003eVerrucomicrobiota\u003c/em\u003e are potential biocontrol bacteria. These have been supported by several studies (Zhang et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2022\u003c/span\u003e, Qi et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2024\u003c/span\u003e, Wu et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Many studies have demonstrated that species in the \u003cem\u003eBacilli\u003c/em\u003e, such as \u003cem\u003eBacillus subtilis\u003c/em\u003e and \u003cem\u003eBacillus amyloliquefaciens\u003c/em\u003e, have the ability to control cotton Verticillium wilt (Zhao et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2022\u003c/span\u003e, Liu et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Members of \u003cem\u003eGlomerales\u003c/em\u003e, such as \u003cem\u003eRhizophagus\u003c/em\u003e and \u003cem\u003eGlomus\u003c/em\u003e, have also been reported to alleviate cotton Verticillium wilt (Kobra et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2011\u003c/span\u003e, Orak \u0026amp; Demir, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2011\u003c/span\u003e, Zhang et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). While there are no reports of \u003cem\u003eClostridia\u003c/em\u003e, unclassified \u003cem\u003eBasidiomycota\u003c/em\u003e or \u003cem\u003eBurkholderiales\u003c/em\u003e controlling cotton Verticillium wilt, these groups have been shown to control other plant diseases. For example, some studies have shown that \u003cem\u003eClostridium beijerinckii\u003c/em\u003e in \u003cem\u003eClostridia\u003c/em\u003e can control Fusarium wilt and root-knot nematodes (Ueki et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2017\u003c/span\u003e, Lian et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Certain \u003cem\u003eBasidiomycota\u003c/em\u003e species can control take-all disease in wheat (Gholami et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2019\u003c/span\u003e, Sandargo et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). \u003cem\u003eBurkholderia\u003c/em\u003e, which are common biocontrol agents In \u003cem\u003eBurkholderiales\u003c/em\u003e, can control plant diseases such as rice blast and black rot (Pal et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2022\u003c/span\u003e, Xue et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2022\u003c/span\u003e, Yu et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). There are no reports on the biocontrol effects of \u003cem\u003eArchaeosporales\u003c/em\u003e or \u003cem\u003eVerrucomicrobiota\u003c/em\u003e, but \u003cem\u003eArchaeosporales\u003c/em\u003e is an order of arbuscular mycorrhiza fungi (AMF) (Kruger et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) and \u003cem\u003eVerrucomicrobiota\u003c/em\u003e is one of the main groups of soil prokaryotes (Rakitin et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Both of these groups may play important roles in plant health. The biocontrol bacteria that we screened were mainly \u003cem\u003eBacillus\u003c/em\u003e, with a small amount of \u003cem\u003ePseudomonas\u003c/em\u003e. This may be due to the difficulty in culturing other bacteria. In future studies, culture methods could be optimized to obtain these potential biocontrol bacteria (Lewis et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2021\u003c/span\u003e, Dai et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e"},{"header":"Statements \u0026 Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Tianjin Municipal Science and Technology Program (23YFYSHZ00050 and 24PTLYHZ00300), the Central Guidance Fund for Local Scientific and Technological Development (Establishment and key technologies of agricultural microbial resource library of Xinjiang, 2024YD009), the Xinjiang Agricultural Microorganisms and Fertilizer Creation Key Laboratory Open Project (SYS2024-01) and the Tianjin Synthetic Biotechnology Innovation Capacity Improvement Project (TSBICIP-IJCP-001).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJingjing Wang, Zhiyong Huang, Shiwu Xu, Bo Gao and Wanyi Zhang contributed to the study conception and design. Material preparation, data collection and analysis were performed by Wanyi Zhang, Jingjing Wang Peng Wu, Yan Pang, Shijia Dai, Xiaoxia Zhang and Ying Chen. The first draft of the manuscript was written by Wanyi Zhang and Jingjing Wang and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated during the current study are available in the National Center for Biotechnology Information (NCBI) repository and supplementary materials.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis article does not contain any studies with human participants or animals performed by any of the authors.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eBai H, Feng Z, Zhao L, Feng H, Wei F, Zhou J, Gu A, Zhu H, Peng J \u0026amp; Zhang Y (2022) Efficacy evaluation and mechanism of \u003cem\u003eBacillus subtilis\u003c/em\u003e EBS03 against cotton Verticillium wilt. \u003cem\u003eJournal of Cotton Research\u003c/em\u003e \u003cstrong\u003e5\u003c/strong\u003e: 26.\u003c/li\u003e\n \u003cli\u003eBerg G (2009) Plant-microbe interactions promoting plant growth and health: perspectives for controlled use of microorganisms in agriculture. \u003cem\u003eApplied Microbiology and Biotechnology\u003c/em\u003e \u003cstrong\u003e84\u003c/strong\u003e: 11-18.\u003c/li\u003e\n \u003cli\u003eCarroll CL, Carter CA, Goodhue RE, Lawell CCL \u0026amp; 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Li B (2023) Interactions between \u003cem\u003eVerticillium dahliae\u003c/em\u003e and cotton: pathogenic mechanism and cotton resistance mechanism to Verticillium wilt. \u003cem\u003eFrontiers in Plant Science\u003c/em\u003e \u003cstrong\u003e14\u003c/strong\u003e: 1174281.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"cotton, verticillium wilt, microbial communities, bio-control bacteria, antifungal activity","lastPublishedDoi":"10.21203/rs.3.rs-6884856/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6884856/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eVerticillium wilt is one of the most devastating diseases of cotton. However, effective biocontrol strains are still lacking. The aim of this study is to inform the selection of effective biocontrol strains by comparing the microbiomes of healthy and diseased cotton plants. Our results revealed that \u003cem\u003eVerticillium dahliae \u003c/em\u003eV991 (V991) is the causal agent of cotton Verticillium wilt, significantly altering the bacterial and fungal communities in the roots, rhizosphere and bulk soil. Compared to the diseased cotton in the V991 inoculation group (D), the healthy cotton in the V991 inoculation group (H) and the control cotton in the V991 non-inoculation group (C) both suppressed \u003cem\u003eVerticillium\u003c/em\u003e and \u003cem\u003eFusarium\u003c/em\u003e and enriched taxa of \u003cem\u003eBacilli\u003c/em\u003e, \u003cem\u003eClostridia\u003c/em\u003e, \u003cem\u003eArchacosporales\u003c/em\u003e, \u003cem\u003eGlomerales\u003c/em\u003e, \u003cem\u003eunclassified Basidiomycota \u003c/em\u003eand\u003cem\u003e unclassified Glomeromycota \u003c/em\u003ein the roots, both enriched \u003cem\u003eBurkholderiales \u003c/em\u003ein the rhizosphere soil, both enriched \u003cem\u003eArchaeosporales\u003c/em\u003e and \u003cem\u003eVerrucomicrobiota\u003c/em\u003e in the bulk soil. A total of 20 strains were screened for antagonism to V991, most of them were isolated from the roots of the C group. \u003cem\u003eBacillus amyloliquefaciens \u003c/em\u003eM9 (BM), which was screened from the rhizosphere soil, exhibited the strongest antifungal activity, whereas \u003cem\u003eBacillus cereus\u003c/em\u003e R19 (BR), which was screened from the root, exhibited weaker antifungal activity. Pot experiments showed that the application of BR and BM (10\u003csup\u003e9\u003c/sup\u003e CFU/mL) reduced the disease incidence by 44.44% and 33.33%, respectively, compared to the control. Field experiments showed that BR reduced the disease incidence by 88.46%, while BM reduced it by 50.01%. These results demonstrate the effectiveness of comparative microbiome analysis in guiding the selection of highly effective biocontrol strains.\u003c/p\u003e","manuscriptTitle":"Development of biocontrol agents for cotton verticillium wilt using microbiome analysis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-18 11:20:12","doi":"10.21203/rs.3.rs-6884856/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"594b4b1a-60b5-4e0c-9ed0-d34d80c407d2","owner":[],"postedDate":"June 18th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-10-31T11:23:26+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-18 11:20:12","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6884856","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6884856","identity":"rs-6884856","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}