Bacillus megaterium controls melon Fusarium wilt disease through its effects on keystone soil taxa

Bacillus megaterium controls melon Fusarium wilt disease through its effects on keystone soil taxa | 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 Bacillus megaterium controls melon Fusarium wilt disease through its effects on keystone soil taxa Xiujun Lu, Qiingmei Li, Bowen Li, Fang Liu, Yeqing Wang, Wenshuo Ning, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4443184/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 01 Dec, 2025 Read the published version in Plant and Soil → Version 1 posted 6 You are reading this latest preprint version Abstract Aims Melon Fusarium wilt, caused by Fusarium. oxysporum f. sp. melonis , is a severe soil-borne disease that reduces melon yield. Biological control approaches have been shown to be effective for the control of melon Fusarium wilt and could contribute to the sustainable development of the melon industry. Bacillus megaterium (BM) is a biocontrol strain that has been shown to promote plant growth and control plant diseases. However, few studies have examined the mechanism by which BM controls melon wilt disease. Methods In this study, we investigated the effect of BM on the growth of melon plants, as well as on soil microbial communities, the soil microbial co-occurrence network, and keystone soil taxa. Results Using a pot experiment, we showed that the incidence of melon Fusarium wilt decreased from 68.33% (CK, inoculated with sterile water) to 26.67% (inoculated with BM), and the control efficiency was 60.00%. In the field experiment, the incidence of melon Fusarium wilt was reduced from 5.56% (naturally occurring) to 1.67% after BM treatment, and the control efficiency was 69.44%. BM treatment also promoted the growth of melon plants and increased the yield of melon to 20.35%. The abundance of potentially beneficial microbes (e.g., Flavobacterium , Nocardioides , Streptomyces , and Chaetomium ) and potentially pathogenic microbes (e.g., Alternaria , Aspergillus , Mortierella , and Plectosphaerella ) was higher and lower in the BM treatment than in the CK, respectively. Co-occurrence network complexity was higher in the BM treatment than in the CK, and the keystone taxa OTU2869 ( Pseudomonas ), OTU3763 ( Sphingobacterium ), and OTU2440 ( Streptomyces ) play key roles in the BM treatment than in the CK. Conclusions The results of our study indicated that BM could be an effective biocontrol agent for the control of Fusarium wilt that could increase melon yield. BM also altered the composition of keystone soil taxa, indicating that it could alter the composition of the soil microbial community, which could promote plant growth and decrease the incidence of melon Fusarium wilt. Bacillus megaterium melon Fusarium wilt soil microbial community co-occurrence network melon yield Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction Melon Fusarium wilt (MFW), caused by the Fusarium. oxysporum f. sp. melonis , is a severe soil-borne disease that has deleterious effects on melon plants (Gava, 2016 ). MFW severely affects the growth of melon plants during the entire reproductive period, and it ultimately reduces yields and hinders the sustainable development of the melon industry. Chemical fungicides are commonly used for the control of MFW (Bharath et al., 2006 ). However, the intensive use of chemical fungicides can have negative environmental and human health effects (Peres, 2020; Rong, 2020). Biological control is widely used for the prevention and control of soil-borne diseases for its broad-spectrum, persistent, and environmentally friendly characteristics (Grosch et al., 2012 ; Whipps, 2001 ), and is considered one of the most promising and safe approaches for crop pest and disease management (Han et al., 2019 ). Many antagonistic microorganisms, such as Bacillus spp., Trichoderma spp., and Pseudomonas fluorescens , have been reported to protect melon plants from attack by Fusarium oxysporum f. sp. melonis (Gava, 2016 ; Özaktan and Bora, 2000 ; Zhang et al., 2020). Bacillus megaterium has multiple plant growth-promoting traits, including the ability to solubilize phosphorus (P) and suppress major phytopathogens (Chinnaswamy et al., 2018 ; Zhao et al., 2021 ). Previous studies have reported that the secondary metabolite iturin from B. megaterium has a strong inhibitory effect on potato late blight ( Phytophthora infestans ) (Wang et al., 2020 ) and rice blast ( Magnaporthe oryzae ) (Liao et al., 2014 ). However, the effects of B. megaterium on MFW remain unclear. Soil microorganisms are an important component of soil ecosystems, and they play a key role in enhancing ecosystem multifunctionally (Bar-On et al., 2018 ; Yang et al., 2023 ). The use of appropriate agronomic management practices can improve soil health by regulating the abundance of beneficial microbes (Hartman et al., 2018 ) and enhancing ecosystem multifunctionality (Yang et al., 2023 ). The application of biological control agents can promote plant growth, maintain soil–plant system health by optimizing the soil flora, and recruit beneficial microorganisms to improve plant stress resistance and control soil-borne diseases (Shi et al., 2017 ; Wu et al., 2016 ). Brevibacillus laterosporus has been shown to reduce the incidence of potato common scab disease by increasing the abundance of beneficial microbes, such as Bacillus and Pseudomonas , and reducing the abundance of Streptomyces scabies (Chen et al., 2017 ). B. megaterium enhances soil microbial community composition and soil nutrient bioavailability, which promotes the growth of cucumber (Zhao et al., 2021 ). However, the response of the soil microbial community to B. megaterium in melon systems has not yet been characterized. Keystone taxa play key roles in agricultural systems by enhancing the responses of plants to pathogens, improving soil multifunctionality, and promoting organic matter decomposition (Mendes et al., 2014; Ze et al., 2013; Li et al., 2023 ; Banerjee et al. 2018). The effect of B. megaterium treatment on keystone taxa in melon soil has not yet been clarified. In this study, we explored the effect of B. megaterium on MFW, plant growth, soil microbial community composition, and keystone taxa through pot and field experiments. Specifically, our aims were to 1) investigate the effects of B. megaterium on the growth and yield of melon, soil microbial community composition, and keystone soil taxa and 2) evaluate the efficacy of B. megaterium for the control of MFW. 2. Materials and methods 2.1 Pot experimental design The pot experiments were performed in a greenhouse (16 h:8 h light:dark photoperiod at 25 ± 1°C) at Hebei Agriculture University. Soils for the pot experiment were collected from the site of the field experiment before fertilization in December 2019. The soils were passed through a 2 mm sieve and mixed homogeneously with B. megaterium fermentation product (2×10 5 CFU/ml) prior to conducting the pot experiment. The same volume of sterilized water was used as a control. The pot experiment was performed using a randomized complete block design with three replicates for each treatment, and each replicate comprised 10 polypropylene pots filled with 3 kg of dry soil. Two melon seeds were planted per pot. 2.1.1 Growth analysis During the flowering and fruiting period of melon, the root length, aboveground and belowground biomass, total plant dry weight, and root/shoot ratio of melon plants from five randomly selected pots in each replicate were measured. 2.1.2 MFW incidence After 90 days since melons were planted, dead melon seedlings were noted, and the incidence of MFW (%) in each treatment was calculated to evaluate the efficacy of B. megaterium for controlling MFW. 2.2 Field experimental design The field experiment [ B. megaterium (BM) and clean water as the control (CK)] was conducted at a vegetable planting cooperative (Qingxian, Hebei Province, China), where melon has been continuously cropped for 11 years, from January to May 2020 using a completely randomized block design with three replicate plots per treatment, and the area of each replicate plot was 33.6 m 2 . The amount of B. megaterium agent (2×10 8 CFU/ml) applied was 75 L/hm 2 , and B. megaterium was applied dropwise around the roots of melon plants after diluting it 500 times with clean water. 2.2.1 Growth and yield analysis Plant height and the number of leaves were recorded 10 days after melon plant colonization; melon yield was determined at the harvest stage. After melon harvest, the roots and shoots of plants were sampled and then dried at 105°C for 30 min and at 75°C until a constant weight was achieved. 2.2.2 Fusarium wilt disease analysis The total number of plants in each plot was counted after melon plant colonization, and the number of dead melon seedlings from the flowering and fruiting period to the harvest period was determined. The incidence of MFW (%) and the efficacy of B. megaterium for the control of MFW in each treatment were calculated. 2.2.3 Soil sampling and analysis of soil properties Ten days after melons were planted, five samples (2.5 cm in diameter) were taken and pooled in each replicate plot. Each sample was thoroughly mixed, placed through a 2 mm sieve, and separated into two equal parts; the first part was stored at -80℃ for soil DNA extraction, and the second part was air-dried at room temperature to determine the content of available P (AP), the content of available potassium (AK), and pH. DNA extraction and high-throughput sequencing were performed by Majorbio Bio-Pharm Technology Co., Ltd. (Shanghai, China). The content of soil AP, the content of soil AK, and pH were determined following the methods of Bao ( 2000 ). 2.3 Statistical analysis Student’s t -test was used to determine the effects of B. megaterium treatment on plant growth, MFW incidence, melon yield, AP, AK, pH, the abundance of potentially beneficial and pathogenic microbes, and the inhibitory activity and spore germination of Fusarium oxysporum f. sp. melonis ; the threshold for statistical significance was P < 0.05. Pearson correlation coefficients were used to determine the associations among soil microbes, soil chemical properties, plant indicators, yield, and MFW. SPSS 18.0 software was used to conduct statistical analyses. Results were expressed as mean ± SD (n = 3). Principal coordinate analysis (PCoA) was performed using genus-level data and binary Jaccard distances to determine the significance of differences in the structure of soil microbial communities among samples. The high-throughput sequencing data were used to conduct co-occurrence network analysis of the bacterial and fungal communities at the OTU level. A correlation matrix was created in R using the “psych” package, and a co-occurrence network was constructed using Gephi (version 0.9.2) (Li et al., 2023 ). Spearman correlations between OTUs were analyzed, and significant associations were inferred using the following criteria: |r| > 0.6 and P < 0.05. Keystone taxa in the networks were identified using the threshold values of Zi and Pi . Nodes spread in module hubs ( Pi 2.5), connectors ( Pi > 0.62, Zi 0.62, Zi > 2.5) were considered to represent generalists that might play key roles in the microbial community as predicted by network theory. 3. Results 3.1 Effect of B. megaterium on defense against MFW BM treatment efficiently suppressed MFW (Table 1 ). MFW incidence was significantly lower in the BM treatment than in the CK in both pot and field experiments, and the control efficiency of Fusarium wilt was 60.00% and 69.44%, respectively. This result indicates that BM alleviated the symptoms of MFW, suggesting that it is effective for the control of MFW. Table 1 Suppression of Bacillus megaterium on fusarium wilt of melon Treatments Pot experiment Field experiment Disease incidence (%) Control efficiency (%) Disease incidence (%) Control efficiency (%) CK 68.33 ± 5.77a --- 5.56 ± 0.96a --- BM 26.67 ± 16.07b 60.00 ± 25.88 1.67 ± 0.00b 69.44 ± 4.81 Note: BM, Bacillus megaterium ; CK, control with water. Different letters in the same columns indicate significant differences among different treatments at the P < 0.05 level. 3.2 Effect of B. megaterium on the agronomic characters and yield of melon BM treatment promoted plant growth and increased melon yield (Fig. 1 ). In the pot experiment, total plant dry weight, root dry weight, root length, and root/shoot ratio were 65.74%, 112.50%, 49.27%, and 16.67% higher, respectively, in the BM treatment than in the CK, and these differences were significant (Fig. 1 A– 1 D) ( P < 0.05). In the field experiment, the root dry weight was markedly higher in the BM treatment than in the CK (Fig. 1 G) ( P < 0.05). Plant height and leaf number were higher in the BM treatment than in the CK, but no significant differences were observed between the BM treatment and the CK (Fig. 1 E, 1 F). Melon yield was 20.35% higher in the BM treatment than in the CK; however, this difference was not significant (Fig. 1 H). 3.3 Effect of B. megaterium on soil chemical properties BM treatment improved the chemical properties of melon soils (Fig. 2 ). The content of AP and AK was 12.54% and 6.75% higher in the BM treatment than in the CK (Fig. 2 A, 2 B), and the pH was lower in the BM treatment than in the CK (Fig. 2 C). However, no significant differences in AP, AK, and pH were observed between the BM treatment and the CK. 3.4 Effect of B. megaterium on soil microbial community composition BM treatment altered the structure of the soil microbial community (Fig. 3 ). In the PCoA based on the binary Jaccard distance for the bacterial community, the first principal coordinate (PC1) and second principal coordinate (PC2) explained 25.06% and 22.86% of the total variance, respectively. The BM treatment was separated from the CK along PC1 (ANOSIM R = 0.4074, P = 0.098) (Fig. 3 A). For the fungal community, PC1 and PC2 explained 28.92% and 25.45% of the total variance, respectively. The fungal communities in soil from the BM treatment were also separated from those of the CK along PC1 (ANOSIM R = 0.4815, P = 0.098) (Fig. 3 C). The BM treatment had a significant effect on the relative abundance of both bacterial and fungal phyla (Fig. 3 ). The five most abundant bacterial phyla in all samples were Proteobacteria, Actinobacteriota, Bacteroidota, Firmicutes, and Chloroflexi. The relative abundances of Actinobacteriota and Chloroflexi were higher in the BM treatment than in the CK, and the relative abundances of Proteobacteria and Bacteroidota were lower in the BM treatment than in the CK (Fig. 3 B). The relative abundance of Ascomycota was higher in the BM treatment than in the CK, and the relative abundance of Mortierellomycota was lower in the BM treatment than in the CK (Fig. 3 D). We analyzed specific bacterial and fungal genera known to be related to plant growth and health. The relative abundances of potentially beneficial and pathogenic microbes in melon soil were altered by BM treatment (Fig. 4 ). The abundance of potentially beneficial microbes, such as Flavobacterium , Nocardioides , Streptomyces , and Chaetomium , was higher in the BM treatment than in the CK (Fig. 4 A– 4 D), and the abundance of Streptomyces and Chaetomium in the BM and CK treatments significantly differed ( P < 0.05). The abundance of potentially pathogenic microbes, such as Alternaria , Aspergillus , Mortierella , and Plectosphaerella , was significantly lower in the BM treatment than in the CK, and these differences were significant, with the exception of Alternaria (Fig. 4 E– 4 H) ( P < 0.05). 3.5 Effects of B. megaterium on soil microbial co-occurrence networks Co-occurrence networks were analyzed at the OTU level to clarify microbial interactions and differences between soil fungal and bacterial communities in soils in the BM treatment (Fig. 5 ). The number of edges, average degree, network density, and average clustering coefficient in the bacterial and fungal co-occurrence networks were higher in the BM treatment than in the CK (Fig. 5 , Table S1 ). These results indicate that the complexity of both the bacterial and fungal networks was higher in the BM treatment than in the CK (Fig. 5 ). In addition, the BM treatment induced changes in the keystone taxa (Fig. 6 , Table S2, Table S3). In the bacterial networks, two nodes (OTU1928 and OUT3229) were classified as module hubs in the CK network, and three nodes (OTU2390, OTU1252, and OUT3208) were classified as module hubs in the BM network (Fig. 6 A). OTU2438 (RB41, genus level), OTU2730 (RB41), OTU2390 (RB41), OTU1872 ( Pedobacter ), and OTU2619 ( Sporocytophaga ) had high degrees in the CK network, and OTU374 ( Nordella , genus level), OTU2868 ( Subgroup _10), OTU2869 ( Pseudomonas ), OTU3763 ( Sphingobacterium ), and OTU2440 ( Streptomyces ) had high degrees in the BM network (Table S2). The connector proportion was higher in the BM treatment (57.14%) than in the CK (54.55%) in the fungal network (Fig. 6 B). OTU228 ( Papulaspora , genus level), OTU241 ( Cephaliophora ), OTU346 ( Mortierella ), OTU329 ( Chrysosporium ), and OTU335 ( Geomyces ) had high degrees in the CK network, and OTU298 ( Leucothecium , genus level), OTU52 ( Acremonium ), OTU147 ( Preussia ), OTU273 ( Ascobolus ), and OTU313 ( Neocosmospora ) had high degrees in the BM network (Table S3). 3.6 Effects of biotic and abiotic factors on melon yield and MFW Spearman correlations were used to clarify the relationships of biotic and abiotic factors with yield and MFW (Fig. 7 ). AP and AK were positively correlated with leaf number, plant height, root dry weight, and melon yield. The abundance of potentially beneficial microbes, such as Streptomyces , Chaetomium , Flavobacterium , and Nocardioides , was positively associated with leaf number, plant height, and melon yield. The abundance of potentially phytopathogenic microbes, such as Alternaria , Mortierella , and Plectosphaerella , was negatively correlated with leaf number, root dry weight, and melon yield, respectively. Moreover, AP, AK, leaf number, plant height, and root dry weight were negatively associated with the incidence of MFW, and the abundance of Mortierella and Plectosphaerella was positively associated with the incidence of MFW. 4. Discussion The effectiveness of using biological control agents to control plant pathogens has been extensively studied, given that they do not generate environmental pollution like pesticides (Lin et al., 2018 ). Bacillus spp. is widely used to control plant diseases and promote plant growth; they are often considered important biocontrol agents in agricultural production (Han et al., 2019 ; Hu et al., 2021 ; Yang et al., 2022 ). There is thus a need to clarify the mechanisms underlying the efficacy of biological control agents for controlling plant diseases, as this has implications for the control of soil-borne pathogens in agricultural systems. The application of BM promoted the growth of melon plants. The total plant dry weight, root dry weight, root length, root/shoot ratio, and melon yield were all higher in the BM treatment than in the CK in both the pot and field experiments. This finding is consistent with the results of previous studies indicating that B. megaterium application increases the shoot and root biomass of cucumber plants and cucumber yield (Zhao et al., 2021 ); this probably stems from the ability of B. megaterium to increase the bioavailability of P and K, which promotes melon plant growth (Zhao et al., 2021 ). We found that BM treatment increased the content of soil AP and AK, which promoted the absorption and utilization of nutrients by plants, and this led to increases in melon yield. Correlation analysis indicated that both AP and AK were significantly and positively associated with plant height, leaf number, root dry weight, and melon yield and negatively associated with MFW incidence. In addition, Bacillus spp. can produce secondary metabolites that promote plant growth. Previous studies have found that iturin A and fengycin A produced by B. megaterium WL-3 enhance plant photosynthetic efficiency, plant growth, and potato yield (Wang et al., 2020 ). This might explain why the BM treatment promoted plant growth and increased melon yield. BM treatment inhibited the development of MFW caused by Fusarium oxysporum f. sp. melonis . This result was consistent with the findings of previous studies showing that B. megaterium can mitigate the deleterious effects of pathogen infection in the field and promote plant growth (Ahmed et al., 2021 ; Yang et al., 2022 ). Previous studies have reported that BM can produce a variety of antimicrobial compounds, such as bacillomycin D, fengycin, and iturin (Jasim et al., 2016 ; Ma et al., 2016 ; Wang et al., 2020 ), which can suppress plant pathogens in vitro and control plant diseases (Jin et al., 2020 ; Liao. T., 2014; Wang et al., 2020 ). We found that B. megaterium inhibited the growth and the spore germination of Fusarium oxysporum f. sp. melonis (Table S4). We also found that BM contained the ituA and fenB genes (Table S5, Fig S1 ), which show strong antimicrobial activity against pathogens, such as Fusarium oxysporum , Aspergillus flavus , and Fusarium graminearum (Liu et al., 2020 ). The secondary metabolites of B. megaterium might promote melon plant growth and suppress melon plant diseases (Gu et al., 2017 ; Zhou et al., 2019 ). Microbial communities play key roles in maintaining plant health and promoting plant growth. In this study, PCoA revealed significant differences in the composition of bacterial and fungal communities between BM and CK treatments. This indicated that BM induced changes in the structure of the bacterial and fungal communities. These results are consistent with the findings of previous studies that have examined the structure of bacterial and fungal communities in cucumber and potato plants following the application of a different B. megaterium strain and S treptomyces pactum (Li et al., 2019 ; Zhao et al., 2021 ). Actinobacteriota and Chloroflexi were more abundant in soil in the BM treatment than in the CK. The abundance of Streptomyces and Chaetomium was significantly higher in the BM treatment than in the CK; some species in these genera are antagonistic to plant pathogens. Li et al. ( 2019 ) found that S treptomyces pactum Act12 reduced the severity of yellow leaf curl virus disease and promoted plant growth in tomato. Chaetomium species can promote cucumber growth and protect plants from cucumber crown rot disease (Sabet et al., 2013 ). In our study, both Streptomyces and Chaetomium were negatively associated with the incidence of MFW and positively associated with plant indicators, such as leaf number, plant height, root dry weight, and melon yield. The abundance of some pathogenic microbes, such as Aspergillus , Mortierella , and Plectosphaerella , was lower in the BM treatment than in the CK. Aspergillus flavus can produce carcinogenic toxins called aflatoxins, which are associated with food and feed safety hazards (Amaike and Keller, 2011). Mortierella comprises pathogenic fungi that can cause plant root rot disease (Os GJV, 2001 ). Xu et al. ( 2014 ) found that Plectosphaerella cucumerina isolated from tomato plants can cause plant wilting and root rot. Previous studies have reported that B. megaterium has a strong inhibitory effect on multiple strains of pathogenic bacteria, such as Aspergillus and Alternaria Nees (Mannaa and Kim, 2018 ; Vásconez et al., 2020 ). In our study, the abundance of Streptomyces and Chaetomium was positively related to leaf number, plant height, and melon yield, and the abundance of Mortierella and Plectosphaerella was negatively associated with leaf number, plant height, root dry weight, and melon yield and positively related to MFW incidence. BM treatment improved the composition of the soil microbial community, increased the abundance of potentially beneficial microbes, reduced the abundance of potentially pathogenic microbes, and enhanced soil health, which might explain why B. megaterium inhibited MFW and promoted melon yield. Co-occurrence network analysis was performed to clarify the interactions among soil fungal and bacterial communities under B. megaterium treatment. The number of edges of both bacterial and fungal networks was higher in BM soil than in CK soil, indicating that the abundance of microbial taxa potentially involved in microbial interactions was higher in the BM treatment than in the CK. The average degree, network density, and average clustering coefficient in the bacterial and fungal networks were higher in the BM treatment than in the CK, which indicated that the bacterial and fungal networks were more complex in the BM treatment than in the CK. Nodes distributed in connectors and module hubs were considered keystone taxa in networks. In this study, we detected the bacterial keystone taxa OTU3208 ( Paenibacillus ), OTU2869 ( Pseudomonas ), OTU3763 ( Sphingobacterium ), and TU2440 ( Streptomyces ) in BM soils. Members of Paenibacillus , Pseudomonas , and Streptomyces can control watermelon wilt disease, avocado white root rot, and potato late blight (Ling et al., 2011 ; Sandra et al., 2020 ; Fu et al., 2022 ). Members of Paenibacillus and Sphingobacterium can promote plant growth and decompose organic pollutants (Brian et al., 2004; Li et al., 2007 ). We found that keystone taxa in the fungal networks, such as OTU298 ( Leucothecium ) and OTU147 ( Preussia ), play key roles in BM soils. Members of Leucothecium can inhibit the growth of mosquito larvae (Gupta et al., 1999). The endophytic fungus Preussia sp. can produce spiropreussione A, which has been shown to inhibit human cancers (Chen et al., 2013 ). In the CK network, keystone taxa such as OTU346 ( Mortierella ) can cause plant root rot disease (Os GJV, 2001 ). Thus, these keystone taxa in the BM treatment might contribute to improvements in the soil environment and the development of disease-suppressive soil after the long-term continuous cropping of melon. 5. Conclusions In summary, we found that the application of B. megaterium effectively inhibits MFW caused by Fusarium oxysporum f. sp. melonis , promotes melon plant growth, and increases melon yield. The increases in potentially beneficial bacteria and fungi and changes in keystone taxa under B. megaterium treatment can stimulate plant growth and development and enhance soil health. Decreases in potentially pathogenic microbes following B. megaterium treatment have the potential to mitigate MFW, which can promote melon growth and ultimately increase melon yield. The use of B. megaterium can thus enhance soil health and promote plant growth in continuous melon cropping systems. These findings indicate that the application of B. megaterium can inhibit MFW, improve soil microbial community composition, promote melon plant growth and yield, and promote the sustainable and healthy development of the melon industry. 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Appl Soil Ecol 103:1–12 Yang W, Zhao YN, Yang Y, Zhang MS, Mao XX, Guo YJ, Li XY, Tao B, Qi YZ, Ma L, Liu WJ, Li BW, Di HJ (2022) A Genomic Analysis of HT517 Reveals the Genetic Basis of Its Abilities to Promote Growth and Control Disease in Greenhouse Tomato. Int J Genomics 2022 Xu J, Xu X, Wang L, Jiang Y, Zhang W, Cao Y (2014) Biological characteristics of tomato wilt fungus. J Shenyang Aricultural Univ 6:673–678 Yang X, Hu H-W, Yang G-W, Cui Z-L, Chen Y-L (2023) Crop rotational diversity enhances soil microbiome network complexity and multifunctionality. Geoderma 436. Zhao Y, Mao X, Zhang M, Yang W, Di HJ, Ma L, Liu W, Li BJA, Ecosystems E (2021) The application of Bacillus Megaterium alters soil microbial community composition, bioavailability of soil phosphorus and potassium, and cucumber growth in the plastic shed system of North China. Agriculture, Ecosystems & Environment 307, 107236 Zhou H, Zou Q, Hu L, Zhu H, Ren Z, Liu E (2019) Isolation and identification of Bacillus tequilensis JN-369 and antimicrobial substance analysis. Chin J Pesticide Sci 21:52–58 Supplementary Files SupplementalMaterial.docx Cite Share Download PDF Status: Published Journal Publication published 01 Dec, 2025 Read the published version in Plant and Soil → Version 1 posted Editorial decision: Major revisions 03 Jul, 2024 Reviewers agreed at journal 24 May, 2024 Reviewers invited by journal 21 May, 2024 Editor invited by journal 20 May, 2024 Editor assigned by journal 20 May, 2024 First submitted to journal 20 May, 2024 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. 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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-4443184","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":305174933,"identity":"dceb1ca5-2744-4a56-bd82-bd15c88d4e95","order_by":0,"name":"Xiujun Lu","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Xiujun","middleName":"","lastName":"Lu","suffix":""},{"id":305174934,"identity":"add91646-44f3-49bf-be7b-e8f7521da7f6","order_by":1,"name":"Qiingmei Li","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAu0lEQVRIiWNgGAWjYBACPmYgkVBhI8fPzHz4AVFa2EBaHpxJM5ZsZ0szIE4LEDM+bDucuOE8j4IEcVrYmbduSGxLS9x8mIfBgKHGJpoIh7GV3Ug4Z2O87TDvgQcMx9JyGwhr4TG7kVCWJrvtMF+CAWPDYWK1sB1m3NzMYyBBgpa2w4obmInXAvILMJAlDgMDOYEYv/DzH9528wcoKvsPH37wocaGsBYgQIrABCKUo2kZBaNgFIyCUYANAAC3ij2SaU8GgwAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0002-9941-2173","institution":"Hebei Agricultural University","correspondingAuthor":true,"prefix":"","firstName":"Qiingmei","middleName":"","lastName":"Li","suffix":""},{"id":305174935,"identity":"5ba0f362-ae9a-4326-8374-5159d58061c2","order_by":2,"name":"Bowen Li","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Bowen","middleName":"","lastName":"Li","suffix":""},{"id":305174936,"identity":"044a0736-5956-49f6-82a5-08ce1e2f8e54","order_by":3,"name":"Fang Liu","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Fang","middleName":"","lastName":"Liu","suffix":""},{"id":305174937,"identity":"3d69506f-37b2-48c6-bbf8-0309ea22a8c7","order_by":4,"name":"Yeqing Wang","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Yeqing","middleName":"","lastName":"Wang","suffix":""},{"id":305174938,"identity":"bf28f80f-2f52-40ec-9594-fe0a9945180f","order_by":5,"name":"Wenshuo Ning","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Wenshuo","middleName":"","lastName":"Ning","suffix":""},{"id":305174939,"identity":"d0b32867-cba5-4404-a1df-2ef0cb8e0aca","order_by":6,"name":"Yanan Liu","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Yanan","middleName":"","lastName":"Liu","suffix":""},{"id":305174940,"identity":"0a2d1058-ce53-45ec-8b96-a8c9e6de1a63","order_by":7,"name":"Hongbo Zhao","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Hongbo","middleName":"","lastName":"Zhao","suffix":""}],"badges":[],"createdAt":"2024-05-19 06:28:35","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4443184/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4443184/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s11104-025-07914-5","type":"published","date":"2025-12-01T15:57:43+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":57642224,"identity":"8d3d3c62-8a83-458a-a942-26d31d51d116","added_by":"auto","created_at":"2024-06-03 17:52:17","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1161846,"visible":true,"origin":"","legend":"\u003cp\u003eGrowth promoting effect of \u003cem\u003eBacillus megaterium\u003c/em\u003e on melon plants in both pot experiment (A-D) and field experiment (E-H).\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4443184/v1/8f18ee52142732831a37f6df.jpeg"},{"id":57642227,"identity":"edeeefdc-079c-4e91-9aee-acc597031c8e","added_by":"auto","created_at":"2024-06-03 17:52:17","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1064246,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of \u003cem\u003eBacillus megaterium\u003c/em\u003e on the content of available phosphorus (A), available potassium (B), and pH (C) in soil.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4443184/v1/ba6ac7519290204d04e702fe.jpeg"},{"id":57642226,"identity":"95b819ca-366b-4eba-8f00-8b21cd80240d","added_by":"auto","created_at":"2024-06-03 17:52:17","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":310825,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of \u003cem\u003eBacillus megaterium\u003c/em\u003e on soil bacterial (A, B) and fungi (C, D) composition.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4443184/v1/a16d33b3880546b575891ab7.jpeg"},{"id":57642225,"identity":"be79aade-5f40-41f1-8039-20afa9a1d234","added_by":"auto","created_at":"2024-06-03 17:52:17","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1436170,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of \u003cem\u003eBacillus megaterium\u003c/em\u003e on the abundance of potential plant beneficial (A-D) and phytopathogenic (E-H) microbes.\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4443184/v1/cedebf33982029ab2eda54c1.jpeg"},{"id":57642228,"identity":"648b6b13-59d1-4d25-b450-bd4f7a0e2007","added_by":"auto","created_at":"2024-06-03 17:52:17","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2594215,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of \u003cem\u003eBacillus megaterium \u003c/em\u003efermentation broth on the hyphae morphology (40×) (A) and spore germination (B) of \u003cem\u003eFusarium oxysporum\u003c/em\u003e f. sp. \u003cem\u003emelonis.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4443184/v1/9d07110170ae073f60e93225.jpeg"},{"id":57642605,"identity":"070ab3b3-bf76-45fd-886a-03b0ce44e2d6","added_by":"auto","created_at":"2024-06-03 18:00:18","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1345981,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of \u003cem\u003eBacillus megaterium \u003c/em\u003ecrude lipopeptide extract on the inhibitory activity (A) and hyphae morphology (40×) (B) of \u003cem\u003eFusarium oxysporum\u003c/em\u003e f. sp. \u003cem\u003emelonis.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4443184/v1/d01a653a8f21666abcaad6bf.jpeg"},{"id":57642229,"identity":"7fc91824-5d9c-4949-9f3c-94e548eaeed6","added_by":"auto","created_at":"2024-06-03 17:52:17","extension":"jpeg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":329759,"visible":true,"origin":"","legend":"\u003cp\u003ePCR amplification of \u003cem\u003eBacillus megaterium\u003c/em\u003e lipopeptide antibiotics.\u003c/p\u003e","description":"","filename":"floatimage7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4443184/v1/9fbe537ea8b0406d5a250027.jpeg"},{"id":97724836,"identity":"2a90ae81-d52a-43a9-b9b2-f9f2ec95e03a","added_by":"auto","created_at":"2025-12-08 16:13:46","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":18172625,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4443184/v1/d688b381-12ec-4302-ac2f-2b482b5a7814.pdf"},{"id":57642230,"identity":"843e04da-eba6-452c-b7a9-e96e985fc1db","added_by":"auto","created_at":"2024-06-03 17:52:18","extension":"docx","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":72994,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalMaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-4443184/v1/77940b92de19bbafcfd981a0.docx"}],"financialInterests":"","formattedTitle":"Bacillus megaterium controls melon Fusarium wilt disease through its effects on keystone soil taxa","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eMelon Fusarium wilt (MFW), caused by the \u003cem\u003eFusarium. oxysporum\u003c/em\u003e f. sp. \u003cem\u003emelonis\u003c/em\u003e, is a severe soil-borne disease that has deleterious effects on melon plants (Gava, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). MFW severely affects the growth of melon plants during the entire reproductive period, and it ultimately reduces yields and hinders the sustainable development of the melon industry. Chemical fungicides are commonly used for the control of MFW (Bharath et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). However, the intensive use of chemical fungicides can have negative environmental and human health effects (Peres, 2020; Rong, 2020). Biological control is widely used for the prevention and control of soil-borne diseases for its broad-spectrum, persistent, and environmentally friendly characteristics (Grosch et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Whipps, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2001\u003c/span\u003e), and is considered one of the most promising and safe approaches for crop pest and disease management (Han et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eMany antagonistic microorganisms, such as \u003cem\u003eBacillus\u003c/em\u003e spp., \u003cem\u003eTrichoderma\u003c/em\u003e spp., and \u003cem\u003ePseudomonas fluorescens\u003c/em\u003e, have been reported to protect melon plants from attack by \u003cem\u003eFusarium oxysporum\u003c/em\u003e f. sp. \u003cem\u003emelonis\u003c/em\u003e (Gava, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; \u0026Ouml;zaktan and Bora, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Zhang et al., 2020). \u003cem\u003eBacillus megaterium\u003c/em\u003e has multiple plant growth-promoting traits, including the ability to solubilize phosphorus (P) and suppress major phytopathogens (Chinnaswamy et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Zhao et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Previous studies have reported that the secondary metabolite iturin from \u003cem\u003eB. megaterium\u003c/em\u003e has a strong inhibitory effect on potato late blight (\u003cem\u003ePhytophthora infestans\u003c/em\u003e) (Wang et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) and rice blast (\u003cem\u003eMagnaporthe oryzae\u003c/em\u003e) (Liao et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). However, the effects of \u003cem\u003eB. megaterium\u003c/em\u003e on MFW remain unclear.\u003c/p\u003e \u003cp\u003eSoil microorganisms are an important component of soil ecosystems, and they play a key role in enhancing ecosystem multifunctionally (Bar-On et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Yang et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The use of appropriate agronomic management practices can improve soil health by regulating the abundance of beneficial microbes (Hartman et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) and enhancing ecosystem multifunctionality (Yang et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The application of biological control agents can promote plant growth, maintain soil\u0026ndash;plant system health by optimizing the soil flora, and recruit beneficial microorganisms to improve plant stress resistance and control soil-borne diseases (Shi et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Wu et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). \u003cem\u003eBrevibacillus laterosporus\u003c/em\u003e has been shown to reduce the incidence of potato common scab disease by increasing the abundance of beneficial microbes, such as \u003cem\u003eBacillus\u003c/em\u003e and \u003cem\u003ePseudomonas\u003c/em\u003e, and reducing the abundance of \u003cem\u003eStreptomyces scabies\u003c/em\u003e (Chen et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). \u003cem\u003eB. megaterium\u003c/em\u003e enhances soil microbial community composition and soil nutrient bioavailability, which promotes the growth of cucumber (Zhao et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). However, the response of the soil microbial community to \u003cem\u003eB. megaterium\u003c/em\u003e in melon systems has not yet been characterized. Keystone taxa play key roles in agricultural systems by enhancing the responses of plants to pathogens, improving soil multifunctionality, and promoting organic matter decomposition (Mendes et al., 2014; Ze et al., 2013; Li et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Banerjee et al. 2018). The effect of \u003cem\u003eB. megaterium\u003c/em\u003e treatment on keystone taxa in melon soil has not yet been clarified.\u003c/p\u003e \u003cp\u003eIn this study, we explored the effect of \u003cem\u003eB. megaterium\u003c/em\u003e on MFW, plant growth, soil microbial community composition, and keystone taxa through pot and field experiments. Specifically, our aims were to 1) investigate the effects of \u003cem\u003eB. megaterium\u003c/em\u003e on the growth and yield of melon, soil microbial community composition, and keystone soil taxa and 2) evaluate the efficacy of \u003cem\u003eB. megaterium\u003c/em\u003e for the control of MFW.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Pot experimental design\u003c/h2\u003e \u003cp\u003eThe pot experiments were performed in a greenhouse (16 h:8 h light:dark photoperiod at 25\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C) at Hebei Agriculture University. Soils for the pot experiment were collected from the site of the field experiment before fertilization in December 2019. The soils were passed through a 2 mm sieve and mixed homogeneously with \u003cem\u003eB. megaterium\u003c/em\u003e fermentation product (2\u0026times;10\u003csup\u003e5\u003c/sup\u003e CFU/ml) prior to conducting the pot experiment. The same volume of sterilized water was used as a control. The pot experiment was performed using a randomized complete block design with three replicates for each treatment, and each replicate comprised 10 polypropylene pots filled with 3 kg of dry soil. Two melon seeds were planted per pot.\u003c/p\u003e \u003cdiv id=\"Sec4\" class=\"Section3\"\u003e \u003ch2\u003e2.1.1 Growth analysis\u003c/h2\u003e \u003cp\u003eDuring the flowering and fruiting period of melon, the root length, aboveground and belowground biomass, total plant dry weight, and root/shoot ratio of melon plants from five randomly selected pots in each replicate were measured.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section3\"\u003e \u003ch2\u003e2.1.2 MFW incidence\u003c/h2\u003e \u003cp\u003eAfter 90 days since melons were planted, dead melon seedlings were noted, and the incidence of MFW (%) in each treatment was calculated to evaluate the efficacy of \u003cem\u003eB. megaterium\u003c/em\u003e for controlling MFW.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Field experimental design\u003c/h2\u003e \u003cp\u003eThe field experiment [\u003cem\u003eB. megaterium\u003c/em\u003e (BM) and clean water as the control (CK)] was conducted at a vegetable planting cooperative (Qingxian, Hebei Province, China), where melon has been continuously cropped for 11 years, from January to May 2020 using a completely randomized block design with three replicate plots per treatment, and the area of each replicate plot was 33.6 m\u003csup\u003e2\u003c/sup\u003e. The amount of \u003cem\u003eB. megaterium\u003c/em\u003e agent (2\u0026times;10\u003csup\u003e8\u003c/sup\u003e CFU/ml) applied was 75 L/hm\u003csup\u003e2\u003c/sup\u003e, and \u003cem\u003eB. megaterium\u003c/em\u003e was applied dropwise around the roots of melon plants after diluting it 500 times with clean water.\u003c/p\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003e2.2.1 Growth and yield analysis\u003c/h2\u003e \u003cp\u003ePlant height and the number of leaves were recorded 10 days after melon plant colonization; melon yield was determined at the harvest stage. After melon harvest, the roots and shoots of plants were sampled and then dried at 105\u0026deg;C for 30 min and at 75\u0026deg;C until a constant weight was achieved.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003e2.2.2 Fusarium wilt disease analysis\u003c/h2\u003e \u003cp\u003eThe total number of plants in each plot was counted after melon plant colonization, and the number of dead melon seedlings from the flowering and fruiting period to the harvest period was determined. The incidence of MFW (%) and the efficacy of \u003cem\u003eB. megaterium\u003c/em\u003e for the control of MFW in each treatment were calculated.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003e2.2.3 Soil sampling and analysis of soil properties\u003c/h2\u003e \u003cp\u003eTen days after melons were planted, five samples (2.5 cm in diameter) were taken and pooled in each replicate plot. Each sample was thoroughly mixed, placed through a 2 mm sieve, and separated into two equal parts; the first part was stored at -80℃ for soil DNA extraction, and the second part was air-dried at room temperature to determine the content of available P (AP), the content of available potassium (AK), and pH. DNA extraction and high-throughput sequencing were performed by Majorbio Bio-Pharm Technology Co., Ltd. (Shanghai, China). The content of soil AP, the content of soil AK, and pH were determined following the methods of Bao (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2000\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Statistical analysis\u003c/h2\u003e \u003cp\u003eStudent\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-test was used to determine the effects of \u003cem\u003eB. megaterium\u003c/em\u003e treatment on plant growth, MFW incidence, melon yield, AP, AK, pH, the abundance of potentially beneficial and pathogenic microbes, and the inhibitory activity and spore germination of \u003cem\u003eFusarium oxysporum\u003c/em\u003e f. sp. \u003cem\u003emelonis\u003c/em\u003e; the threshold for statistical significance was \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05. Pearson correlation coefficients were used to determine the associations among soil microbes, soil chemical properties, plant indicators, yield, and MFW. SPSS 18.0 software was used to conduct statistical analyses. Results were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD (n\u0026thinsp;=\u0026thinsp;3). Principal coordinate analysis (PCoA) was performed using genus-level data and binary Jaccard distances to determine the significance of differences in the structure of soil microbial communities among samples. The high-throughput sequencing data were used to conduct co-occurrence network analysis of the bacterial and fungal communities at the OTU level. A correlation matrix was created in R using the \u0026ldquo;psych\u0026rdquo; package, and a co-occurrence network was constructed using Gephi (version 0.9.2) (Li et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Spearman correlations between OTUs were analyzed, and significant associations were inferred using the following criteria: |r| \u0026gt; 0.6 and \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05. Keystone taxa in the networks were identified using the threshold values of \u003cem\u003eZi\u003c/em\u003e and \u003cem\u003ePi\u003c/em\u003e. Nodes spread in module hubs (\u003cem\u003ePi\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.62, \u003cem\u003eZi\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;2.5), connectors (\u003cem\u003ePi\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.62, \u003cem\u003eZi\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;2.5), and network hubs (\u003cem\u003ePi\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.62, \u003cem\u003eZi\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;2.5) were considered to represent generalists that might play key roles in the microbial community as predicted by network theory.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Effect of \u003cem\u003eB. megaterium\u003c/em\u003e on defense against MFW\u003c/h2\u003e \u003cp\u003eBM treatment efficiently suppressed MFW (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). MFW incidence was significantly lower in the BM treatment than in the CK in both pot and field experiments, and the control efficiency of Fusarium wilt was 60.00% and 69.44%, respectively. This result indicates that BM alleviated the symptoms of MFW, suggesting that it is effective for the control of MFW.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSuppression of \u003cem\u003eBacillus megaterium\u003c/em\u003e on fusarium wilt of melon\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eTreatments\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003ePot experiment\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eField experiment\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDisease incidence (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eControl efficiency (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDisease incidence (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eControl efficiency (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCK\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e68.33\u0026thinsp;\u0026plusmn;\u0026thinsp;5.77a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e---\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5.56\u0026thinsp;\u0026plusmn;\u0026thinsp;0.96a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e---\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e26.67\u0026thinsp;\u0026plusmn;\u0026thinsp;16.07b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e60.00\u0026thinsp;\u0026plusmn;\u0026thinsp;25.88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e69.44\u0026thinsp;\u0026plusmn;\u0026thinsp;4.81\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003eNote: BM, \u003cem\u003eBacillus megaterium\u003c/em\u003e; CK, control with water. Different letters in the same columns indicate significant differences among different treatments at the \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 level.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Effect of \u003cem\u003eB. megaterium\u003c/em\u003e on the agronomic characters and yield of melon\u003c/h2\u003e \u003cp\u003eBM treatment promoted plant growth and increased melon yield (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e1\u003c/span\u003e). In the pot experiment, total plant dry weight, root dry weight, root length, and root/shoot ratio were 65.74%, 112.50%, 49.27%, and 16.67% higher, respectively, in the BM treatment than in the CK, and these differences were significant (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e1\u003c/span\u003eA\u0026ndash;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e1\u003c/span\u003eD) (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). In the field experiment, the root dry weight was markedly higher in the BM treatment than in the CK (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e1\u003c/span\u003eG) (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Plant height and leaf number were higher in the BM treatment than in the CK, but no significant differences were observed between the BM treatment and the CK (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e1\u003c/span\u003eE, \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). Melon yield was 20.35% higher in the BM treatment than in the CK; however, this difference was not significant (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e1\u003c/span\u003eH).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Effect of \u003cem\u003eB. megaterium\u003c/em\u003e on soil chemical properties\u003c/h2\u003e \u003cp\u003eBM treatment improved the chemical properties of melon soils (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The content of AP and AK was 12.54% and 6.75% higher in the BM treatment than in the CK (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, \u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e2\u003c/span\u003eB), and the pH was lower in the BM treatment than in the CK (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). However, no significant differences in AP, AK, and pH were observed between the BM treatment and the CK.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Effect of \u003cem\u003eB. megaterium\u003c/em\u003e on soil microbial community composition\u003c/h2\u003e \u003cp\u003eBM treatment altered the structure of the soil microbial community (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e3\u003c/span\u003e). In the PCoA based on the binary Jaccard distance for the bacterial community, the first principal coordinate (PC1) and second principal coordinate (PC2) explained 25.06% and 22.86% of the total variance, respectively. The BM treatment was separated from the CK along PC1 (ANOSIM R\u0026thinsp;=\u0026thinsp;0.4074, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.098) (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). For the fungal community, PC1 and PC2 explained 28.92% and 25.45% of the total variance, respectively. The fungal communities in soil from the BM treatment were also separated from those of the CK along PC1 (ANOSIM R\u0026thinsp;=\u0026thinsp;0.4815, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.098) (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e3\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003eThe BM treatment had a significant effect on the relative abundance of both bacterial and fungal phyla (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The five most abundant bacterial phyla in all samples were Proteobacteria, Actinobacteriota, Bacteroidota, Firmicutes, and Chloroflexi. The relative abundances of Actinobacteriota and Chloroflexi were higher in the BM treatment than in the CK, and the relative abundances of Proteobacteria and Bacteroidota were lower in the BM treatment than in the CK (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). The relative abundance of Ascomycota was higher in the BM treatment than in the CK, and the relative abundance of Mortierellomycota was lower in the BM treatment than in the CK (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e3\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003eWe analyzed specific bacterial and fungal genera known to be related to plant growth and health. The relative abundances of potentially beneficial and pathogenic microbes in melon soil were altered by BM treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The abundance of potentially beneficial microbes, such as \u003cem\u003eFlavobacterium\u003c/em\u003e, \u003cem\u003eNocardioides\u003c/em\u003e, \u003cem\u003eStreptomyces\u003c/em\u003e, and \u003cem\u003eChaetomium\u003c/em\u003e, was higher in the BM treatment than in the CK (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e4\u003c/span\u003eA\u0026ndash;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e4\u003c/span\u003eD), and the abundance of \u003cem\u003eStreptomyces\u003c/em\u003e and \u003cem\u003eChaetomium\u003c/em\u003e in the BM and CK treatments significantly differed (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The abundance of potentially pathogenic microbes, such as \u003cem\u003eAlternaria\u003c/em\u003e, \u003cem\u003eAspergillus\u003c/em\u003e, \u003cem\u003eMortierella\u003c/em\u003e, and \u003cem\u003ePlectosphaerella\u003c/em\u003e, was significantly lower in the BM treatment than in the CK, and these differences were significant, with the exception of \u003cem\u003eAlternaria\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e4\u003c/span\u003eE\u0026ndash;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e4\u003c/span\u003eH) (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Effects of \u003cem\u003eB. megaterium\u003c/em\u003e on soil microbial co-occurrence networks\u003c/h2\u003e \u003cp\u003eCo-occurrence networks were analyzed at the OTU level to clarify microbial interactions and differences between soil fungal and bacterial communities in soils in the BM treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The number of edges, average degree, network density, and average clustering coefficient in the bacterial and fungal co-occurrence networks were higher in the BM treatment than in the CK (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e5\u003c/span\u003e, Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). These results indicate that the complexity of both the bacterial and fungal networks was higher in the BM treatment than in the CK (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e5\u003c/span\u003e). In addition, the BM treatment induced changes in the keystone taxa (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e6\u003c/span\u003e, Table S2, Table S3). In the bacterial networks, two nodes (OTU1928 and OUT3229) were classified as module hubs in the CK network, and three nodes (OTU2390, OTU1252, and OUT3208) were classified as module hubs in the BM network (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). OTU2438 (RB41, genus level), OTU2730 (RB41), OTU2390 (RB41), OTU1872 (\u003cem\u003ePedobacter\u003c/em\u003e), and OTU2619 (\u003cem\u003eSporocytophaga\u003c/em\u003e) had high degrees in the CK network, and OTU374 (\u003cem\u003eNordella\u003c/em\u003e, genus level), OTU2868 (\u003cem\u003eSubgroup\u003c/em\u003e_10), OTU2869 (\u003cem\u003ePseudomonas\u003c/em\u003e), OTU3763 (\u003cem\u003eSphingobacterium\u003c/em\u003e), and OTU2440 (\u003cem\u003eStreptomyces\u003c/em\u003e) had high degrees in the BM network (Table S2). The connector proportion was higher in the BM treatment (57.14%) than in the CK (54.55%) in the fungal network (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). OTU228 (\u003cem\u003ePapulaspora\u003c/em\u003e, genus level), OTU241 (\u003cem\u003eCephaliophora\u003c/em\u003e), OTU346 (\u003cem\u003eMortierella\u003c/em\u003e), OTU329 (\u003cem\u003eChrysosporium\u003c/em\u003e), and OTU335 (\u003cem\u003eGeomyces\u003c/em\u003e) had high degrees in the CK network, and OTU298 (\u003cem\u003eLeucothecium\u003c/em\u003e, genus level), OTU52 (\u003cem\u003eAcremonium\u003c/em\u003e), OTU147 (\u003cem\u003ePreussia\u003c/em\u003e), OTU273 (\u003cem\u003eAscobolus\u003c/em\u003e), and OTU313 (\u003cem\u003eNeocosmospora\u003c/em\u003e) had high degrees in the BM network (Table S3).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.6 Effects of biotic and abiotic factors on melon yield and MFW\u003c/h2\u003e \u003cp\u003eSpearman correlations were used to clarify the relationships of biotic and abiotic factors with yield and MFW (Fig.\u0026nbsp;\u003cspan refid=\"Fig14\" class=\"InternalRef\"\u003e7\u003c/span\u003e). AP and AK were positively correlated with leaf number, plant height, root dry weight, and melon yield. The abundance of potentially beneficial microbes, such as \u003cem\u003eStreptomyces\u003c/em\u003e, \u003cem\u003eChaetomium\u003c/em\u003e, \u003cem\u003eFlavobacterium\u003c/em\u003e, and \u003cem\u003eNocardioides\u003c/em\u003e, was positively associated with leaf number, plant height, and melon yield. The abundance of potentially phytopathogenic microbes, such as \u003cem\u003eAlternaria\u003c/em\u003e, \u003cem\u003eMortierella\u003c/em\u003e, and \u003cem\u003ePlectosphaerella\u003c/em\u003e, was negatively correlated with leaf number, root dry weight, and melon yield, respectively. Moreover, AP, AK, leaf number, plant height, and root dry weight were negatively associated with the incidence of MFW, and the abundance of \u003cem\u003eMortierella\u003c/em\u003e and \u003cem\u003ePlectosphaerella\u003c/em\u003e was positively associated with the incidence of MFW.\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThe effectiveness of using biological control agents to control plant pathogens has been extensively studied, given that they do not generate environmental pollution like pesticides (Lin et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). \u003cem\u003eBacillus\u003c/em\u003e spp. is widely used to control plant diseases and promote plant growth; they are often considered important biocontrol agents in agricultural production (Han et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Hu et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Yang et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). There is thus a need to clarify the mechanisms underlying the efficacy of biological control agents for controlling plant diseases, as this has implications for the control of soil-borne pathogens in agricultural systems.\u003c/p\u003e \u003cp\u003eThe application of BM promoted the growth of melon plants. The total plant dry weight, root dry weight, root length, root/shoot ratio, and melon yield were all higher in the BM treatment than in the CK in both the pot and field experiments. This finding is consistent with the results of previous studies indicating that \u003cem\u003eB. megaterium\u003c/em\u003e application increases the shoot and root biomass of cucumber plants and cucumber yield (Zhao et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2021\u003c/span\u003e); this probably stems from the ability of \u003cem\u003eB. megaterium\u003c/em\u003e to increase the bioavailability of P and K, which promotes melon plant growth (Zhao et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). We found that BM treatment increased the content of soil AP and AK, which promoted the absorption and utilization of nutrients by plants, and this led to increases in melon yield. Correlation analysis indicated that both AP and AK were significantly and positively associated with plant height, leaf number, root dry weight, and melon yield and negatively associated with MFW incidence. In addition, \u003cem\u003eBacillus\u003c/em\u003e spp. can produce secondary metabolites that promote plant growth. Previous studies have found that iturin A and fengycin A produced by \u003cem\u003eB. megaterium\u003c/em\u003e WL-3 enhance plant photosynthetic efficiency, plant growth, and potato yield (Wang et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). This might explain why the BM treatment promoted plant growth and increased melon yield. BM treatment inhibited the development of MFW caused by \u003cem\u003eFusarium oxysporum\u003c/em\u003e f. sp. \u003cem\u003emelonis\u003c/em\u003e. This result was consistent with the findings of previous studies showing that \u003cem\u003eB. megaterium\u003c/em\u003e can mitigate the deleterious effects of pathogen infection in the field and promote plant growth (Ahmed et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Yang et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Previous studies have reported that BM can produce a variety of antimicrobial compounds, such as bacillomycin D, fengycin, and iturin (Jasim et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Ma et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Wang et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), which can suppress plant pathogens \u003cem\u003ein vitro\u003c/em\u003e and control plant diseases (Jin et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Liao. T., 2014; Wang et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). We found that \u003cem\u003eB. megaterium\u003c/em\u003e inhibited the growth and the spore germination of \u003cem\u003eFusarium oxysporum\u003c/em\u003e f. sp. \u003cem\u003emelonis\u003c/em\u003e (Table S4). We also found that BM contained the \u003cem\u003eituA\u003c/em\u003e and \u003cem\u003efenB\u003c/em\u003e genes (Table S5, Fig \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e), which show strong antimicrobial activity against pathogens, such as \u003cem\u003eFusarium oxysporum\u003c/em\u003e, \u003cem\u003eAspergillus flavus\u003c/em\u003e, and \u003cem\u003eFusarium graminearum\u003c/em\u003e (Liu et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The secondary metabolites of \u003cem\u003eB. megaterium\u003c/em\u003e might promote melon plant growth and suppress melon plant diseases (Gu et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Zhou et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eMicrobial communities play key roles in maintaining plant health and promoting plant growth. In this study, PCoA revealed significant differences in the composition of bacterial and fungal communities between BM and CK treatments. This indicated that BM induced changes in the structure of the bacterial and fungal communities. These results are consistent with the findings of previous studies that have examined the structure of bacterial and fungal communities in cucumber and potato plants following the application of a different \u003cem\u003eB. megaterium\u003c/em\u003e strain and S\u003cem\u003etreptomyces pactum\u003c/em\u003e (Li et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Zhao et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). \u003cem\u003eActinobacteriota\u003c/em\u003e and \u003cem\u003eChloroflexi\u003c/em\u003e were more abundant in soil in the BM treatment than in the CK. The abundance of \u003cem\u003eStreptomyces\u003c/em\u003e and \u003cem\u003eChaetomium\u003c/em\u003e was significantly higher in the BM treatment than in the CK; some species in these genera are antagonistic to plant pathogens. Li et al. (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) found that S\u003cem\u003etreptomyces pactum\u003c/em\u003e Act12 reduced the severity of yellow leaf curl virus disease and promoted plant growth in tomato. \u003cem\u003eChaetomium\u003c/em\u003e species can promote cucumber growth and protect plants from cucumber crown rot disease (Sabet et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). In our study, both \u003cem\u003eStreptomyces\u003c/em\u003e and \u003cem\u003eChaetomium\u003c/em\u003e were negatively associated with the incidence of MFW and positively associated with plant indicators, such as leaf number, plant height, root dry weight, and melon yield. The abundance of some pathogenic microbes, such as \u003cem\u003eAspergillus\u003c/em\u003e, \u003cem\u003eMortierella\u003c/em\u003e, and \u003cem\u003ePlectosphaerella\u003c/em\u003e, was lower in the BM treatment than in the CK. \u003cem\u003eAspergillus flavus\u003c/em\u003e can produce carcinogenic toxins called aflatoxins, which are associated with food and feed safety hazards (Amaike and Keller, 2011). \u003cem\u003eMortierella\u003c/em\u003e comprises pathogenic fungi that can cause plant root rot disease (Os GJV, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). Xu et al. (\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) found that \u003cem\u003ePlectosphaerella cucumerina\u003c/em\u003e isolated from tomato plants can cause plant wilting and root rot. Previous studies have reported that \u003cem\u003eB. megaterium\u003c/em\u003e has a strong inhibitory effect on multiple strains of pathogenic bacteria, such as \u003cem\u003eAspergillus\u003c/em\u003e and \u003cem\u003eAlternaria\u003c/em\u003e Nees (Mannaa and Kim, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; V\u0026aacute;sconez et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In our study, the abundance of \u003cem\u003eStreptomyces\u003c/em\u003e and \u003cem\u003eChaetomium\u003c/em\u003e was positively related to leaf number, plant height, and melon yield, and the abundance of \u003cem\u003eMortierella\u003c/em\u003e and \u003cem\u003ePlectosphaerella\u003c/em\u003e was negatively associated with leaf number, plant height, root dry weight, and melon yield and positively related to MFW incidence. BM treatment improved the composition of the soil microbial community, increased the abundance of potentially beneficial microbes, reduced the abundance of potentially pathogenic microbes, and enhanced soil health, which might explain why \u003cem\u003eB. megaterium\u003c/em\u003e inhibited MFW and promoted melon yield.\u003c/p\u003e \u003cp\u003eCo-occurrence network analysis was performed to clarify the interactions among soil fungal and bacterial communities under \u003cem\u003eB. megaterium\u003c/em\u003e treatment. The number of edges of both bacterial and fungal networks was higher in BM soil than in CK soil, indicating that the abundance of microbial taxa potentially involved in microbial interactions was higher in the BM treatment than in the CK. The average degree, network density, and average clustering coefficient in the bacterial and fungal networks were higher in the BM treatment than in the CK, which indicated that the bacterial and fungal networks were more complex in the BM treatment than in the CK. Nodes distributed in connectors and module hubs were considered keystone taxa in networks. In this study, we detected the bacterial keystone taxa OTU3208 (\u003cem\u003ePaenibacillus\u003c/em\u003e), OTU2869 (\u003cem\u003ePseudomonas\u003c/em\u003e), OTU3763 (\u003cem\u003eSphingobacterium\u003c/em\u003e), and TU2440 (\u003cem\u003eStreptomyces\u003c/em\u003e) in BM soils. Members of \u003cem\u003ePaenibacillus\u003c/em\u003e, \u003cem\u003ePseudomonas\u003c/em\u003e, and \u003cem\u003eStreptomyces\u003c/em\u003e can control watermelon wilt disease, avocado white root rot, and potato late blight (Ling et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Sandra et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Fu et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Members of \u003cem\u003ePaenibacillus\u003c/em\u003e and \u003cem\u003eSphingobacterium\u003c/em\u003e can promote plant growth and decompose organic pollutants (Brian et al., 2004; Li et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). We found that keystone taxa in the fungal networks, such as OTU298 (\u003cem\u003eLeucothecium\u003c/em\u003e) and OTU147 (\u003cem\u003ePreussia\u003c/em\u003e), play key roles in BM soils. Members of \u003cem\u003eLeucothecium\u003c/em\u003e can inhibit the growth of mosquito larvae (Gupta et al., 1999). The endophytic fungus \u003cem\u003ePreussia\u003c/em\u003e sp. can produce spiropreussione A, which has been shown to inhibit human cancers (Chen et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). In the CK network, keystone taxa such as OTU346 (\u003cem\u003eMortierella\u003c/em\u003e) can cause plant root rot disease (Os GJV, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). Thus, these keystone taxa in the BM treatment might contribute to improvements in the soil environment and the development of disease-suppressive soil after the long-term continuous cropping of melon.\u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eIn summary, we found that the application of \u003cem\u003eB. megaterium\u003c/em\u003e effectively inhibits MFW caused by \u003cem\u003eFusarium oxysporum\u003c/em\u003e f. sp. \u003cem\u003emelonis\u003c/em\u003e, promotes melon plant growth, and increases melon yield. The increases in potentially beneficial bacteria and fungi and changes in keystone taxa under \u003cem\u003eB. megaterium\u003c/em\u003e treatment can stimulate plant growth and development and enhance soil health. Decreases in potentially pathogenic microbes following \u003cem\u003eB. megaterium\u003c/em\u003e treatment have the potential to mitigate MFW, which can promote melon growth and ultimately increase melon yield. The use of \u003cem\u003eB. megaterium\u003c/em\u003e can thus enhance soil health and promote plant growth in continuous melon cropping systems. These findings indicate that the application of \u003cem\u003eB. megaterium\u003c/em\u003e can inhibit MFW, improve soil microbial community composition, promote melon plant growth and yield, and promote the sustainable and healthy development of the melon industry.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eConflict of interest\u003c/h2\u003e \u003cp\u003eThis manuscript is approved by all authors for publication and has no conflict of interest.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis research was supported by National Key R\u0026amp;D Program of China (2022YFD1901301-3), Hebei Province Agricultural Industry System Project (HBCT2024130204), Hebei Natural Science Youth Foundation (C2023204233), the earmarked fund for CARS-13, S\u0026amp;T Program of Hebei (20326812D, 21326905D).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAhmed HFA, Seleiman MF, Al-Saif AM, Alshiekheid MA, Battaglia ML, Taha RS (2021) Biological Control of Celery Powdery Mildew Disease Caused by DC In Vitro and In Vivo Conditions. 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Chin J Pesticide Sci 21:52\u0026ndash;58\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"plant-and-soil","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"plso","sideBox":"Learn more about [Plant and Soil](https://www.springer.com/journal/11104)","snPcode":"11104","submissionUrl":"https://submission.nature.com/new-submission/11104/3","title":"Plant and Soil","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Bacillus megaterium, melon Fusarium wilt, soil microbial community, co-occurrence network, melon yield","lastPublishedDoi":"10.21203/rs.3.rs-4443184/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4443184/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eAims\u003c/h2\u003e \u003cp\u003eMelon Fusarium wilt, caused by \u003cem\u003eFusarium. oxysporum\u003c/em\u003e f. sp. \u003cem\u003emelonis\u003c/em\u003e, is a severe soil-borne disease that reduces melon yield. Biological control approaches have been shown to be effective for the control of melon Fusarium wilt and could contribute to the sustainable development of the melon industry. \u003cem\u003eBacillus megaterium\u003c/em\u003e (BM) is a biocontrol strain that has been shown to promote plant growth and control plant diseases. However, few studies have examined the mechanism by which BM controls melon wilt disease.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eIn this study, we investigated the effect of BM on the growth of melon plants, as well as on soil microbial communities, the soil microbial co-occurrence network, and keystone soil taxa.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eUsing a pot experiment, we showed that the incidence of melon Fusarium wilt decreased from 68.33% (CK, inoculated with sterile water) to 26.67% (inoculated with BM), and the control efficiency was 60.00%. In the field experiment, the incidence of melon Fusarium wilt was reduced from 5.56% (naturally occurring) to 1.67% after BM treatment, and the control efficiency was 69.44%. BM treatment also promoted the growth of melon plants and increased the yield of melon to 20.35%. The abundance of potentially beneficial microbes (e.g., \u003cem\u003eFlavobacterium\u003c/em\u003e, \u003cem\u003eNocardioides\u003c/em\u003e, \u003cem\u003eStreptomyces\u003c/em\u003e, and \u003cem\u003eChaetomium\u003c/em\u003e) and potentially pathogenic microbes (e.g., \u003cem\u003eAlternaria\u003c/em\u003e, \u003cem\u003eAspergillus\u003c/em\u003e, \u003cem\u003eMortierella\u003c/em\u003e, and \u003cem\u003ePlectosphaerella\u003c/em\u003e) was higher and lower in the BM treatment than in the CK, respectively. Co-occurrence network complexity was higher in the BM treatment than in the CK, and the keystone taxa OTU2869 (\u003cem\u003ePseudomonas\u003c/em\u003e), OTU3763 (\u003cem\u003eSphingobacterium\u003c/em\u003e), and OTU2440 (\u003cem\u003eStreptomyces\u003c/em\u003e) play key roles in the BM treatment than in the CK.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eThe results of our study indicated that BM could be an effective biocontrol agent for the control of Fusarium wilt that could increase melon yield. BM also altered the composition of keystone soil taxa, indicating that it could alter the composition of the soil microbial community, which could promote plant growth and decrease the incidence of melon Fusarium wilt.\u003c/p\u003e","manuscriptTitle":"Bacillus megaterium controls melon Fusarium wilt disease through its effects on keystone soil taxa","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-03 17:52:13","doi":"10.21203/rs.3.rs-4443184/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revisions","date":"2024-07-04T01:34:09+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2024-05-24T04:47:09+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-05-21T19:10:15+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"Plant and Soil","date":"2024-05-20T23:29:09+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-05-20T23:22:00+00:00","index":"","fulltext":""},{"type":"submitted","content":"Plant and Soil","date":"2024-05-20T09:59:37+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"plant-and-soil","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"plso","sideBox":"Learn more about [Plant and Soil](https://www.springer.com/journal/11104)","snPcode":"11104","submissionUrl":"https://submission.nature.com/new-submission/11104/3","title":"Plant and Soil","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"ea59b67d-6d5b-4c37-b459-a6498f751855","owner":[],"postedDate":"June 3rd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-12-08T16:11:30+00:00","versionOfRecord":{"articleIdentity":"rs-4443184","link":"https://doi.org/10.1007/s11104-025-07914-5","journal":{"identity":"plant-and-soil","isVorOnly":false,"title":"Plant and Soil"},"publishedOn":"2025-12-01 15:57:43","publishedOnDateReadable":"December 1st, 2025"},"versionCreatedAt":"2024-06-03 17:52:13","video":"","vorDoi":"10.1007/s11104-025-07914-5","vorDoiUrl":"https://doi.org/10.1007/s11104-025-07914-5","workflowStages":[]},"version":"v1","identity":"rs-4443184","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4443184","identity":"rs-4443184","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}