Effect of plant elicitor Isotianil on the rhizosphere soil microbiome composition of banana cultivars with different resistance to Fusarium wilt

Effect of plant elicitor Isotianil on the rhizosphere soil microbiome composition of banana cultivars with different resistance to Fusarium wilt | 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 Effect of plant elicitor Isotianil on the rhizosphere soil microbiome composition of banana cultivars with different resistance to Fusarium wilt Wenlong Zhang, Guangdong Zhou, Ping He, Hongwei Yu, Libo Tian, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5557820/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 13 Jun, 2025 Read the published version in Plant and Soil → Version 1 posted 5 You are reading this latest preprint version Abstract Aims Banana Fusarium wilt disease severely threatens the sustainable development of the banana industry. Regulating the local microbiome to reduce disease incidence offers broader adaptability, with a potential exploration into plant-induced recruitment of beneficial microbes. Our previous study showed that the plant elicitor Isotianil induces resistance to banana Fusarium wilt. This study assessed the impact of Isotianil on soil microbiomes. Methods Pot experiments were conducted with the susceptible banana cultivar Baxijiao and the tolerant cultivar Yunjiao No. 1, inoculating them with the Fusarium pathogen and introducing Isotianil by foliar application and root drenching. Amplicon sequencing was used to analyze the rhizosphere bacterial and fungal communities. Results Results showed that Isotianil had 48% and 55% disease control efficacy rates for Baxijiao and Yunjiao No. 1 after foliar application, and 51% and 55% after root drenching, respectively. Amplicon sequencing revealed that, compared to pathogen alone, foliar application of Isotianil significantly altered the bacterial and fungal community compositions in Yunjiao No. 1, while root drenching significantly changed these communities in both cultivars. Root drenching of Isotianil notably increased the relative abundance of potential beneficial microbes such as Blrii41 , Devosia , and Acremonium and enhanced the stability of bacterial and fungal cross-kingdom networks. Additionally, the application of Isotianil increases the impact of stochastic processes in the bacterial community assembly of Baxijiao while decreasing their importance in Yunjiao No.1. Conclusions These findings suggest that Isotianil application modulates the rhizosphere microbiome, promoting beneficial microbes, stabilizing microbial networks, potentially contributing to disease suppression. Plant health Plant elicitor Rhizosphere 16S and ITS Co-occurrence network Bacterial community assembly Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Soil-borne fungal diseases lead to global annual losses to crop production estimated at 15% (Newbery et al. 2016). Common control measures primarily include breeding resistant varieties, spatial and temporal diversification in planting, chemical control, and biological control (Noble et al. 2005). Although large-scale application of chemical pesticides or fungicides can be highly effective in the short term, the environmental and economic costs are substantial. Growing pathogen resistance to chemical control has been well documented. Consequently, extensive research continues to explore efficient and environmentally friendly control strategies to mitigate biotic stress damage and reduce risks to human health (Ruijven et al. 2020). Plant elicitors as resistance inducers are one option to reduce damage from fungal diseases (Zhou et al. 2023). As small molecules that trigger plant immune responses, elicitors can mimic natural elicitors or defense signaling molecules. They activate defense mechanisms by interacting with their homologous plant receptors or interfering with other defense signaling components (Thakur and Sohal 2013). Synthetic inducers have been successfully applied in crop protection, offering an attractive alternative to traditional biocidal agrochemicals (Bektas and Eulgem 2015). Isotianil, developed by Bayer, is a thiazole-based compound that lacks direct antibacterial or antifungal activity. However, it can activate defense responses against pathogens in different plant species (Ogawa et al. 2011). It has been utilized as a crop protection agent against rice blast and bacterial leaf blight in rice and wheat (Portz et al. 2021). Isotianil induces the accumulation of defense-related genes associated with salicylic acid signaling (e.g., OsWRKY45 ) and defense-related enzymes (e.g., lipoxygenase or PAL) (Braun et al. 2014). Isotianil significantly induces resistance against banana sigatoka leaf spot and lasting up to 28 days (Qi et al. 2019). Moreover, synthetic inducers serve as effective tools in basic research methods, expanding our understanding of plant immunity. The rhizosphere microbiome is crucial for maintaining plant health and productivity (Berendsen et al. 2012; Qu et al. 2020). Plants can protect themselves from different types of stress by assembling a beneficial microbiome through various mechanisms (Trivedi et al. 2020). In response to pathogen infection, plants not only trigger systemic immune responses to directly inhibit pathogen invasion but also modulate the microbiome (Wei et al. 2020). This regulation includes altering the composition and quantity of root exudates, volatiles, and reactive oxygen species, thereby modifying the soil microbiome to directly or indirectly suppress disease occurrence (Wen et al. 2023). The microbial elicitor BAR11 enhances Arabidopsis thaliana resistance to Pseudomonas syringae pv. tomato DC3000 by influencing the accumulation of specific secondary metabolites and altering the composition and diversity of the rhizosphere microbial community, particularly promoting the recruitment of beneficial microorganisms that contribute to enhanced plant defense (Wang et al. 2024). Synthetic elicitors such as methyl salicylic acid and acibenzolar-s-methyl stimulate pine wilt resistance in plants and modulate the root microbiome, increasing the abundance of beneficial microorganisms (Mannaa et al. 2020). Bananas are the world's most traded fruit and the fourth most important food crop, playing a vital role in ensuring the stability and progress of developing countries (Nayar 2010). However, banana production is seriously threatened by Fusarium wilt, caused by Fusarium oxysporum f. sp. cubense ( Foc ), which endangers the sustainable development of the banana industry (Siamak and Zheng 2018). While breeding for disease-resistant varieties has proven to be the most effective control measure, no commercial cultivars with complete immunity to the widely spread Foc Tropic Race 4 (TR4) have been developed (Ploetz 2015). Current control strategies primarily rely on strict quarantine measures, the cultivation of disease-tolerant varieties, and biological control. Traditional fungicides are not feasible because of their high cost and low efficacy. Biological control serves as an advantageous alternative (Pegg et al. 2019). However, the effectiveness of biocontrol agents is often inconsistent (Bardin et al. 2015), especially for soil-borne diseases (Yang et al. 2019; Ptaszek et al. 2023). Therefore, understanding the mechanisms by which bananas recruit beneficial microbes related to disease resistance is crucial for managing Fusarium wilt (Dita et al. 2018). One promising research direction involves enhancing banana resistance through plant immunity elicitors, which may lead to new strategies for recruiting beneficial microbes. This approach not only aids in understanding the complex interactions between bananas and microorganisms but also provides a scientific basis and practical potential for disease management. Our group demonstrated that the application of Isotianil could enhance the expression of genes related to plant resistance induction and starch synthesis in banana corms, indicating its potential to boost the host’s resistance to TR4 (Zhou et al. 2023). However, just how Isotianil impacts the belowground rhizosphere microbiome compositions remain unclear. Considering the significant role of the rhizosphere microbiome in the occurrence and development of Fusarium wilt, this study employed amplicon sequencing to analyze the effects of Isotianil-induced plant resistance on the rhizosphere microbiome of the susceptible banana cultivar Baxijiao and the resistant cultivar Yunjiao No. 1. Materials and methods Varieties and cultivation methods The experiment was conducted using the Fusariam wilt-susceptible Cavendish banana cultivar Baxijiao and the tolerant Cavendish cultivar Yunjiao No. 1. Tissue-cultured plantlets were initially cultivated at 25 °C with a 16 h light / 8 h dark photoperiod for 20 days. After developing 4-5 leaves and establishing roots, the plantlets were transplanted into 5 cm trays filled with a mixture of coir and plantlet substrate (1:1). After 56 days, when approximately 3-4 new leaves had grown, the plantlets were transplanted into 25 cm diameter plastic pots filled with native horticultural red ferrosols soil which has not previously been exposed to bananas and unsterilized banana plantlet substrate (1:1). Pathogen and Isotianil inoculation disease index assessment TR4 is the laboratory-maintained strain 15-1 (Zhang et al. 2018). After being cultured on Potato Dextrose Agar medium at 28 °C for 7 days, spores were collected by washing the plates with sterile water. The spore suspension concentration was determined to be 1×10⁷ spores/mL using a hemocytometer. Isotianil suspension (0.2 g/mL) provided by Bayer Crop Science was applied at the time of transplantation into plastic pots, when the plants had approximately 8 leaves. Each banana plant received a root drench or foliar spray of 100 mL (0.07 mg/mL) Isotianil, applied every 28 days, for a total of three applications. 7 days after the second application of Isotianil, each plant was inoculated with 100 mL of the TR4 spore suspension (1×10⁷ spores/mL) by wounding the roots, while the control treatment was treated with tap water. There were four treatments — CK: control treatment; TA: inoculate TR4 alone; FT: foliar application and inoculate TR4; RT: root drenching and inoculate TR4 — with 45 plants per treatment. Wilt severity was recorded after 62 further days similar as in our previous study (Zhang et al. 2019). Wilt severity was divided into four levels: 0 = healthy plant, 1 = 1 % ‒25 % necrosis in the corm, 2 = 26 % ‒ 50 % necrosis in the corm, 3 = 51% ‒75% necrosis in the corm, 4 = 75 % ‒100 % necrosis in the corm. Disease Index = ∑ (Number of Plants in Each Wilt Severity × Representative Value) / (Total Number of Plants × 4) × 100 . Soil sample collection, DNA extraction, and PCR Banana plants were removed from the soil and vigorously shaken to remove soil around the roots. Each replicate consisted of 3 seedlings, resulting in 15 samples per treatment (A total of 120 samples). The soil adhering to the root surface was then collected using a brush and stored at -80 °C until DNA extraction. Total genomic DNA samples were extracted using the OMEGA Soil DNA Kit (D5625-01) from Omega Bio-Tek, Norcross, GA, USA, according to the protocol provided by the manufacturer. The extracted DNA samples were stored at -20 °C until further analysis. The concentration and purity of the DNA were assessed using a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA), while the integrity was checked via agarose gel electrophoresis. The V3–V4 region of bacterial 16S rRNA genes was amplified using PCR with the forward primer 338F (5'-ACTCCTACGGGAGGCAGCA-3') and the reverse primer 806R (5'-GGACTACHVGGGTWTCTAAT-3'). For fungal community analysis, the ITS1 region of fungal ITS rRNA genes was amplified using the forward primer ITS5F (5'-GGAAGTAAAAGTCGTAACAAGG-3') and the reverse primer ITS1R (5'-GCTGCGTTCTTCATCGATGC-3'). The PCR system and program were referenced from our previous work (Zhang et al. 2024). Sequencing analysis After individual quantification, the amplicons were pooled in equal proportions and sequenced using paired-end 2 × 300 bp reads on an Illumina MiSeq platform, utilizing the MiSeq Reagent Kit v3, at Shanghai Personal Biotechnology Co., Ltd, Shanghai, China. The raw reads were deposited in the NCBI Sequence Read Archive (16S: PRJNA949124; ITS: PRJNA949129). Quality-filtered and merged sequences were denoised using the DADA2 plugin (Callahan et al. 2016) in QIIME2 (Bolyen et al. 2019) to obtain amplicon sequence variants (ASVs). Chloroplast and mitochondrial sequences were removed from all samples. Taxonomic classification of ASVs was performed using the Naive Bayes classifier in QIIME2, based on the SILVA (v132) and UNITE (v8.0) database. To ensure comparability across samples, the sequencing depth was rarefied to 95 % of the minimum sequencing depth (16S: 36525; ITS:65063). The ASV table is provided in Electronic supplementary material 1 – 4 . Data analysis R version 4.1.2 was used for data analysis, with visualization performed using GraphPad Prism 10. Alpha diversity indices of the community were calculated using the vegan package. The vegan package was also employed to calculate differences in community composition between samples using Permutational Multivariate Analysis of Variance (PERMANOVA) and Principal Co-ordinates Analysis (PCoA). Correlation analysis was performed using Spearman correlation. Differences in data between groups were analyzed using the Kruskal-Wallis test, followed by multiple comparison correction using the Benjamini-Hochberg (BH) method in SPSS 25. A cross-kingdom co-occurrence network was constructed using the Hmisc package (R > 0.6, adjusted P < 0.05), with Spearman correlation and BH multiple testing corrections applied. The genus-level networks and indices were visualized and calculated using Gephi 10. A high modularity index and higher proportion of negative correlation edges indicate more stable community network, while a higher average degree represents more complex community network (Gao et al. 2021). The iCAMP, picante and ape package were used to construct phylogenetic-based null model analysis and calculate Beta nearest taxon indice (Beta NTI), the normalized stochasticity ratio (NST) was calculated with the NST package. To ensure the reliability of the randomization process, we performed 1000 randomizations for both Beta NTI and NST. The absolute value of Beta NTI lower than 2 indicates that random processes dominate during community assembly. A higher NST indicates a greater impact of stochasticity processes on community assembly (Ning et al. 2020). Fungal ASVs were assigned into functional guilds using the FUNGuild (Nguyen et al. 2016). Results Incidence of Fusarium wilt in different varieties following TR4 and Isotianil inoculation When inoculated with TR4 alone (TA) the disease index of Fusarium wilt in Yunjiao No. 1 (Disease index: 26.49) was significantly lower than in Baxijiao (51.56). The application of Isotianil (FT: foliar application and inoculate TR4; RT: root drenching and inoculate TR4) significantly reduced the disease index of Fusarium wilt in both Baxijiao (26.61in FT, 25.52 in RT) and Yunjiao No. 1 (11.98 in FT, 11.98 in RT) compared to inoculation with TA. There were no significant differences between RT and FT (Fig. 1) . [Insert Fig. 1] Bacterial and fungal communities’ diversity and composition differences following TR4 and Isotianil inoculation Compared to the control treatment (CK), the TA treatment showed no significant differences in the Chao1 and Shannon indices of bacterial and fungal communities in both Baxijiao and Yunjiao No. 1. Compared to the TA treatment, FT and RT had no significant impact on the Chao1 and Shannon indices of bacterial and fungal communities in both Baxijiao and Yunjiao No.1 (Fig. 2) . [Insert Fig. 2] Compared to the CK, TA significantly changed the bacterial community composition in both Baxijiao and Yunjiao No. 1. Relative to TA, RT significantly changed the bacterial composition in both Baxijiao and Yunjiao No. 1, while FT only significantly affected the bacterial community composition in Yunjiao No. 1. Compared to the CK, TA significantly changed the fungal community composition in Yunjiao No. 1. Compared to TA, both RT and FT significantly changed the fungal composition in Yunjiao No. 1, but had no significant impact on Baxijiao (Fig. 3) . [Insert Fig. 3] Responses of bacterial and fungal taxa following TR4 and Isotianil inoculation For the dominant phylum (relative abundance > 1%), the relative abundance of Firmicutes significantly decreased compared to CK following TA in Baxijiao. Compared to TA, FT significantly decreased the relative abundance of Firmicutes in Baxijiao. RT increased the relative abundance of Ascomycota and decreased the relative abundance of Actinobacteria, Gemmatimonadetes and Rozellomycota in Baxijiao. Compared to CK, TA significantly decreased the relative abundance of Chloroflexi in Yunjiao No. 1. Compared to TA, FT significantly decreased the relative abundance of Firmicutes in Yunjiao No. 1. RT increased the relative abundance of Proteobacteria and Bacteroidetes, while decreased the relative abundance of Chloroflexi, Gemmatimonadetes and Mortierellomycota in Yunjiao No. 1 (Fig. 4) . [Insert Fig. 4] For dominant genera (relative abundance > 1%), compared to CK, TA increased the relative abundance of Fusarium in Baxijiao. Relative to TA, RT increased the relative abundance of Blrii41 , Devosia , and Acremonium , while decreased the relative abundance of Sphingomonas in Baxijiao. Relative to TA, FT significantly increased the relative abundance of Acremonium in Yunjiao No. 1. RT increased the relative abundance of Blrii41 , Devosia , Acremonium and Plectosphaerella , while decreasing the relative abundance of KD4_96 and MND1 in Yunjiao No. 1 (Fig. 5) . In addition, the Blrii41 , Devosia , Acremonium and Plectosphaerella showed a significant negative correlation with disease index in Baxijiao and Yunjiao No. 1, across all TR4-inoculated samples (Table. 1) . [Insert Fig. 5] Table 1 Correlation between dominant bacterial and fungal genera and Fusarium wilt disease index based on Spearman across all TR4-inoculated samples. genus Baxijiao Yunjiao No.1 R P R P Subgroup_6 -0.015 0.705 0.058 0.924 KD4-96 -0.050 0.011 0.374 0.743 A4b -0.316 0.943 0.011 0.034 AD3 0.006 0.447 0.116 0.967 Rokubacteriales 0.103 0.537 0.094 0.502 SBR1031 -0.197 0.144 0.221 0.195 MND1 0.384 0.097 0.250 0.009 Sphingomonas 0.595 0.722 0.054 0.000 BIrii41 -0.551 0.012 -0.370 0.000 Devosia -0.522 0.002 -0.446 0.000 Fusarium -0.078 0.002 -0.446 0.613 Mortierella 0.057 0.619 0.076 0.712 Botryotrichum -0.381 0.602 -0.080 0.010 Gibberella -0.015 0.003 0.428 0.924 Acremonium -0.495 0.000 -0.668 0.001 Didymella 0.308 0.491 0.105 0.040 Plectosphaerella -0.300 0.000 -0.599 0.046 Effects of TR4 and Isotianil inoculation on cross-kingdom co-occurrence networks Compared to CK, TA resulted in decreased network modularity index, proportion of cross-kingdom edges, and average degree in Baxijiao (Fig. 6 a) (Table. 2) . In Yunjiao No. 1, the modularity index and the proportion of negative correlation edges increased (Fig. 6 b) . Relative to TA, both FT and RT increased the modularity index and proportion of negative correlation edges of the network in Baxijiao and Yunjiao No. 1 (Fig. 6 a and b) , but there was no significant change in the average degree (Fig. 6 c and d) . Additionally, compared to CK, TA reduced the proportion of cross-kingdom edges in Baxijiao while increasing it in Yunjiao No. 1, indicating a decrease in the interaction between soil bacteria and fungi in the rhizosphere of Baxijiao and an enhancement in Yunjiao No. 1 (Table. 2) . [Insert Fig. 6] Table 2 The number of edges in cross-kingdom Co-occurrence Networks. Genotype Treatment PFF NFF PBF NBF PBB NBB Baxijiao CK 404 13 101 116 517 367 TA 538 1 95 33 323 259 FT 66 11 186 146 469 259 RT 220 31 242 220 335 266 Yunjiao No.1 CK 446 38 66 70 406 257 TA 161 20 151 124 414 232 FT 161 59 222 207 389 315 RT 112 11 134 132 319 270 Note: PFF: positive correlation edge between fungi and fungi; NFF: negative correlation edge between fungi and fungi. PBF: positive correlation edge between bacteria and fungi. NBF: negative correlation edge between bacteria and fungi. PBB: positive correlation edge between bacteria and bacteria. NBB: negative correlation edge between bacteria and bacteria. Assembly processes of bacterial and fungal communities following TR4 and Isotianil inoculation In the null model, stochasticity processes played a more important role than deterministic ones during the bacterial and fungal community assembly under all treatments (Fig. 7a and Fig. S1a ) . For bacterial community, compared to CK, TA had a higher NST index in Baxijiao, and lower in Yunjiao No.1. Compared to TA, FT and RT significantly reduced the impact of stochasticity processes in Baxijiao bacterial community assembly, while it increased the impact of stochasticity processes in Yunjiao No.1 (Fig. 7b ) . However, in the NST analysis of the fungal community, the NST of the fungal community showed significant deviation, with some values exceeding the expected range of 0 to 1. This suggests that NST analysis may not be applicable to the fungal community in this study (Fig. S1b) . [Insert Fig. 7] FUNGuild functional prediction of fungal community Compared to CK, TA treatment did not result in significant differences in predictions of functional fungal guilds both in Baxijiao and Yunjiao No.1. However, compared to TA, the relative abundance of the Pathogen-Saprotroph-Symbiotroph guild significantly increased in Baxijiao under RT and FT treatments, and significantly increased in Yunjiao No.1 under the RT treatment, the relative abundance of Symbiotroph guild significantly decreased in Yunjiao No.1 under the RT treatment (Table. S1 and S2) . For the plant pathogens within the functional group, no significant differences were observed among treatments (Fig. S2) . Discussion Regulating the soil microbiome to reduce the occurrence of soil-borne diseases has been widely applied in crop production (Gang et al. 2013; Bubici et al. 2019; Fan et al. 2023). With advances in high-throughput sequencing technology, the underlying mechanisms have been further explored and optimized (Zhu et al. 2023). In addition to reducing the abundance of pathogens, research has demonstrated how to increase the number of beneficial microorganisms in the soil through direct inoculation or other measures (Tao et al. 2020). However, in addition to the efficient interaction with target crops, the effectiveness of directly inoculated beneficial microorganisms relies on their ability to successfully colonize the soil. Microorganisms must adapt to the diverse soil environment and overcome the self-regulatory mechanisms of the native microbiome. Consequently, the effectiveness of many high-efficiency microorganisms often remains inconsistent across different field conditions (Chen et al. 2022). By studying the beneficial microbial communities recruited in different resistant varieties, we can better develop effective disease control strategies. Microorganisms actively recruited by plants tend to be more competitive in the soil, enhancing the plant's immune system, promoting healthy growth, and helping to protect against pathogen invasion (Rolfe et al. 2019). Isotianil application enhances the expression of genes related to plant resistance induction and starch synthesis (Zhou et al. 2023). Research on elicitor-induced host resistance has primarily focused on the expression of the plant's own resistance genes (Benhamou 1996; Zhai et al. 2017). However, the soil microbiome has proven to be closely related to disease development. Investigating the soil microbiome will enhance our understanding of disease progression (Wei et al. 2019). In this study, root drenching had a more pronounced impact on soil microorganisms compared to foliar application, likely due to the direct disturbance caused by Isotianil as a carbon source. Foliar application of Isotianil had no significant effect on the bacterial and fungal community composition in Baxijiao. For Yunjiao No.1, on the other hand, both foliar and root applications of Isotianil significantly altered the bacterial and fungal communities. The number of changes in the bacterial and fungal phyla and genera of Yunjiao No.1 was greater than that of Baxijiao. This may be due to more active root exudates or higher rhizosphere metabolic activity in Yunjiao No.1, which consequently altered the abundance of more microbial taxa. Subsequent research could involve analyzing the composition of root exudates after foliar application to link microbial community taxa with the exudates, further confirming the reasons behind this stronger variation. Once these microbes colonize the rhizosphere, they can enhance plant resistance through various mechanisms such as inhibiting pathogens, promoting plant growth, and inducing plant resistance (Kwak et al. 2018; Mendes et al. 2018b). For example, Liu (2023) found that banana varieties' resistance to the TR4 is linked to their ability to recruit Trichoderma and Penicillium , which are stimulated by D-(-)-ribofuranose and propylene glycol. Enhancing soils with these exudates improved resistance in susceptible varieties. Compared to inoculation with TR4 alone, root drenching Isotianil significantly increased the relative abundances of Blrii41 , Devosia , and Acremonium in the rhizosphere of both banana varieties. These microorganisms are all potentially beneficial. Blrii41 may be associated with nutrient transformation and is typically abundant in organic soils (Stephenson and Kahlert 2018; Wei et al. 2024). Devosia and Acremonium have been shown to play important roles in microbe-mediated disease resistance (Grunewaldt-Stöcker and von Alten 2003; Barros et al. 2022). For example, the non-pathogenic strain Pseudomonas syringae DC3000 can reduce the secretion of myristic acid by tomato roots, leading to an increase in the abundance of Devosia in the rhizosphere, which ultimately promotes plant growth and induces the formation of soil-borne disease suppression (Liu et al. 2024). The endophytic fungus Acremonium isolated from Lium davidii has been shown to effectively inhibit various pathogenic fungi and promote plant growth (Khan et al. 2021). Therefore, after the application of Isotianil, the microorganisms recruited in the banana rhizosphere exhibit disease-suppressive potential, which may enhance their ability to resist pathogens. A higher modularity index indicates greater community stability, which is a mark of healthy soil and the recruitment of beneficial microorganisms (Gao et al. 2021; Hassani et al. 2023). The microbial community network in soils resistant to banana wilt had a higher proportion of negative correlations, which may lead to greater stress due to pathogen infection (Lv et al. 2023). In this study, compared to CK, the stability of the microbial community network in Baxijiao decreased after TR4 inoculation, while in Yunjiao No.1, the stability increased. This reflects Yunjiao No.1's stronger microbial regulatory capacity after TR4 infection, shaping more stable network interactions. This phenomenon is also observed in other species. For instance, tobacco varieties resistant to black shank disease ( Phytophthora parasitica ) have more complex and highly connected microbial networks than susceptible varieties (Li et al. 2023). Similarly, common bean varieties resistant to Fusarium wilt possess more complex communities than susceptible varieties (Mendes et al. 2018a). The application of Isotianil improved the stability of the rhizosphere microbiome network, likely by inducing systemic resistance in plants, which enhanced the recruitment of beneficial microorganisms. This elicitor effect is similar to that of biocontrol microorganisms in inducing plant resistance (Cardoni et al. 2023; Ge et al. 2024). Yunjiao No.1 exhibited a stronger response to both TR4 and Isotianil than Baxijiao, and the combination of Yunjiao No.1 with Isotianil demonstrated significant disease resistance potential, which may provide an effective disease control strategy for the banana industry. A higher stochastic process indicates lower environmental selection pressure (Du et al. 2021). After Isotianil application, Baxijiao exhibited a decrease in the Beta nearest taxon index in bacterial communities, while Yunjiao No.1 showed an increase in the Beta nearest taxon index. This suggests that the two banana varieties respond differently to Isotianil. In Baxijiao, the reduced randomness may imply that Isotianil application strengthens its selective control over rhizosphere microorganisms. As environmental selection pressure increases, the microbial community structure becomes more deterministic, which might reflect Baxijiao's strategy to enhance disease resistance by reducing community randomness (Wen et al. 2022). In contrast, Yunjiao No.1, despite showing increased determinism process after TR4 inoculation, experienced greater community stochastic process after Isotianil application, possibly due to a reduced need for strict community assembly control as disease incidence decreased. These differences provide important insights for understanding and improving disease resistance in different banana varieties. Considering cultivar-specific rhizosphere microbial assembly patterns could be a key factor in disease resistance breeding and control strategies. Through this study, we gained insights into how inducers promote the recruitment of beneficial microorganisms by plants, providing a theoretical basis for optimizing soil microbial communities. However, further research is needed to verify the specific roles of these microbial species in maintaining banana health, especially under field conditions. Additionally, metabolomic analysis to identify differences in root exudates could better reveal the mechanisms behind microbial recruitment, helping to identify which beneficial microorganisms have stronger interactions with bananas. By customizing the application of these microbial agents and optimizing soil microbial communities, we can further enhance banana disease resistance. Taking these factors into account, more precise and effective plant disease resistance strategies can be developed, improving crop health and yield, reducing pesticide use, and advancing sustainable agriculture. Conclusions The application of Isotianil not only enhances plant disease resistance but also regulates the stability of the rhizosphere microbiome, promoting the recruitment of beneficial microorganisms. The disease-resistant cultivar Yunjiao No. 1, when combined with Isotianil, demonstrated significant disease resistance effects and beneficial microbiome regulation. These findings provide novel scientific evidence and potential applications for enhancing the disease resistance of banana varieties and improving agricultural practices. Declarations Conflict of interest The authors declare no conflicts of interest. Acknowledgements This work was supported by National Natural Science Foundation of China (NSFC32161143001); Science and Technology Department of Yunnan Provincial Government (202204BI090019), Yunnan Fundamental Research Project (grant No. 202401AT070094) and Yunnan Fundamental Research Project (grant No. 202401BD070001-096) and Yunling Scholar Programme of Yunnan Provincial Government (YNWR-YLXZ-2018-018); Funded in part by Seedqual Initiative of One CGIAR, and the International Atomic Energy Agency, under the project “An Integrative Approach to Enhancing Disease Resistance Against Fusarium Wilt ( Foc TR4) in Banana –Phase II (D23033).” We thank Glenn Hyman from of the Alliance of Bioversity International and CIAT Science Writing Service for his editorial support of this manuscript. Authors’ Contributions Wenlong Zhang: Methodology, Software, Visualization, Writing - review & editing. Guangdong Zhou: Methodology, Visualization. Ping He: Visualization, Supervision, Investigation. Hongwei Yu: Visualization, Software, Investigation. Libo Tian: Visualization, Supervision, Investigation. Yun-Yue Wang: Conceptualization, Supervision. Si-Jun Zheng: Conceptualization, Supervision, Funding acquisition, Writing - review & editing. All authors read and approved the final manuscript. Availability of data and materials Research data have been deposited in the NCBI databases (16S: PRJNA 949124; ITS: PRJNA949129). References Bardin M, Ajouz S, Comby M, Lopez-Ferber M, Graillot B, Siegwart M, Nicot PC (2015). Is the efficacy of biological control against plant diseases likely to be more durable than that of chemical pesticides? Front Plant Sci 6, 566. https://doi.org/10.3389/fpls.2015.00566 Barros FMDR, Pedrinho A, Mendes LW, Freitas CCG, Andreote FD (2022) Interactions between soil bacterial diversity and plant-parasitic nematodes in soybean plants. Appl Environ 88:e00963-22. https://doi.org/10.1128/aem.00963-22 Bektas Y, Eulgem T (2015) Synthetic plant defense elicitors. 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Front Plant Sci 14:1145837. https://doi.org/10.3389/fpls.2023.1145837 Zhu Z, Wu G, Deng R, Hu X, Tan H, Chen Y, Tian Z, Li J (2023) Spatiotemporal biocontrol and rhizosphere microbiome analysis of Fusarium wilt of banana. Commun Biol 6:27. https://doi.org/10.1038/s42003-023-04417-w Supplementary Files SupplementaryMaterial.docx evendepth16s.xls evendepthits.xls filtered16s.xls filteredits.xls Cite Share Download PDF Status: Published Journal Publication published 13 Jun, 2025 Read the published version in Plant and Soil → Version 1 posted Reviewers agreed at journal 21 Apr, 2025 Reviewers invited by journal 21 Apr, 2025 Editor assigned by journal 20 Apr, 2025 First submitted to journal 17 Apr, 2025 Editorial decision: Minor revisions 21 Jan, 2025 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-5557820","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":446016405,"identity":"f5bdd85b-315d-4330-8ce9-7d0a9accc3f0","order_by":0,"name":"Wenlong Zhang","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Wenlong","middleName":"","lastName":"Zhang","suffix":""},{"id":446016406,"identity":"d4e2e3e1-cf91-4f18-b575-c3fcbeb0feaf","order_by":1,"name":"Guangdong Zhou","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Guangdong","middleName":"","lastName":"Zhou","suffix":""},{"id":446016407,"identity":"6853b5b0-bf01-47bf-9215-16bdee701d34","order_by":2,"name":"Ping He","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Ping","middleName":"","lastName":"He","suffix":""},{"id":446016408,"identity":"5573dd9e-b000-444b-98c8-14d4045019e9","order_by":3,"name":"Hongwei Yu","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Hongwei","middleName":"","lastName":"Yu","suffix":""},{"id":446016409,"identity":"845d7fde-8e3b-45a6-8083-bb7a7921940c","order_by":4,"name":"Libo Tian","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Libo","middleName":"","lastName":"Tian","suffix":""},{"id":446016410,"identity":"d37fe6f8-e0d2-4a93-8eff-d0f1beff1d2b","order_by":5,"name":"Yun-Yue Wang","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Yun-Yue","middleName":"","lastName":"Wang","suffix":""},{"id":446016411,"identity":"4ab5ba27-f803-4645-93ba-00627efdece5","order_by":6,"name":"Si-Jun Zheng","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA00lEQVRIiWNgGAWjYHACNhAhx8DA2ECaFmPStSQSr16+gf3Zg487atP7+xc3fq5gsMmXdyCgxeAAQ7rhzDPHc2fceNgseYYhzXLjAUJaGBiOSfO2HcvdIHGwQbKB4bCBISEnyjcwtkn/bTuWbiBxsPknUVoYDjCzSTO21SQY8De2gW2RJ6TD4DAbm2Rv2wHDGTcY2ywbDNIMDAhpkW9vfybxs61Onr//+OObDRU2BvIEHcYMJg8zMEgkMIBDAxiGRIE6BgZ+qFLCtoyCUTAKRsFIAwA2xD9Hy1Xa8QAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0003-1550-3738","institution":"Yunnan Academy of Agricultural Sciences","correspondingAuthor":true,"prefix":"","firstName":"Si-Jun","middleName":"","lastName":"Zheng","suffix":""}],"badges":[],"createdAt":"2024-12-01 10:05:08","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5557820/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5557820/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s11104-025-07569-2","type":"published","date":"2025-06-13T15:57:06+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":81243060,"identity":"4fb7ae79-d4c9-41da-a658-d6cf2e4ee9a5","added_by":"auto","created_at":"2025-04-24 00:17:33","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":21426,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe Fusarium wilt disease index of Baxijiao and Yunjiao No. 1.\u003c/strong\u003e CK: the control treatment; TA: inoculate TR4 alone; FT: foliar application Isotianil and inoculate TR4; RT: root drenching Isotianil and inoculate TR4. Different letters represent significant differences (adjusted \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05) among different treatments within the same variety by Kruskal-Wallis test and BH method.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-5557820/v1/96b478f5d2ac74f7b084e9d6.png"},{"id":81243055,"identity":"0698417d-c4b5-4dba-899b-1cd4c7f70ef3","added_by":"auto","created_at":"2025-04-24 00:17:33","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":99719,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe Alpha diversity of bacterial and fungal communities in Baxijiao (a-b) and Yunjiao No.1(c-d).\u003c/strong\u003e CK: the control treatment; TA: inoculate TR4 alone; FT: foliar application Isotianil and inoculate TR4; RT: root drenching Isotianil and inoculate TR4. Different letters represent significant differences (adjusted \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05) among different treatments within the same variety by Kruskal-Wallis test and BH method.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-5557820/v1/498738866dfe069d4de49804.png"},{"id":81243269,"identity":"b050167c-142b-4c16-a289-6c74fad1fed4","added_by":"auto","created_at":"2025-04-24 00:33:33","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":155823,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe differences in bacterial (a-b) and fungal (c-d)community composition by PERMANOVA and PCoA\u003c/strong\u003e. CK: the control treatment; TA: inoculate TR4 alone; FT: foliar application Isotianil and inoculate TR4; RT: root drenching Isotianil and inoculate TR4.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-5557820/v1/0fb922cb3e3c1042890655c4.png"},{"id":81243061,"identity":"7bfb1b35-b6fe-4ba0-91ca-a464113209bc","added_by":"auto","created_at":"2025-04-24 00:17:33","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":155897,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe relative abundance of dominant bacterial and fungal phyla in Baxijiao (a-b) and Yunjiao No.1 (c-d)\u003c/strong\u003e. CK: the control treatment; TA: inoculate TR4 alone; FT: foliar application Isotianil and inoculate TR4; RT: root drenching Isotianil and inoculate TR4. Different letters represent significant differences (adjusted \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05) by Kruskal-Wallis test and BH method.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-5557820/v1/fc971741ad098a3768980569.png"},{"id":81243065,"identity":"75b0aedc-6a2a-499a-8051-da3a2dd3ed1b","added_by":"auto","created_at":"2025-04-24 00:17:34","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":152236,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe relative abundance of dominant bacterial and fungal genera in Baxijiao (a-b) and Yunjiao No.1(c-d)\u003c/strong\u003e. CK: the control treatment; TA: inoculate TR4 alone; FT: foliar application Isotianil and inoculate TR4; RT: root drenching Isotianil and inoculate TR4. Different letters represent significant differences (adjusted \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05) by Kruskal-Wallis test and BH method.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-5557820/v1/da253c0926de0ceda4c0adda.png"},{"id":81243064,"identity":"209cad5a-8860-4936-af3e-f9c40afee8db","added_by":"auto","created_at":"2025-04-24 00:17:33","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1725186,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCross-kingdom co-occurrence networks of Baxijiao (a) and Yunjiao No.1 (b) and their average degree (c-d).\u003c/strong\u003e The modularity index is located in the bottom right corner of the network. The nodes are colored according to Bacteria and Fungi. Node size represents the degree of connection. Edge color represents positive and negative correlations. CK: the control treatment; TA: inoculate TR4 alone; FT: foliar application Isotianil and inoculate TR4; RT: root drenching Isotianil and inoculate TR4. The significant differences (adjusted \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05) were calculated by Kruskal-Wallis test and BH method.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-5557820/v1/1fc211fb0c5584809c3413d4.png"},{"id":81243254,"identity":"1aa79eb5-c8a2-4ce5-ab5e-b0d98014d3a2","added_by":"auto","created_at":"2025-04-24 00:25:33","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":72442,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAssembly mechanisms of bacterial communities.\u003c/strong\u003e (a) Beta nearest taxon index of different treatments. (b) Normalized stochasticity ratio of different treatments. CK: the control treatment; TA: inoculate TR4 alone; FT: foliar application Isotianil and inoculate TR4; RT: root drenching Isotianil and inoculate TR4. Different letters represent significant differences (adjusted \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05) among different treatments within the same variety by Kruskal-Wallis test and BH method.\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-5557820/v1/de1cbfa3b00b08f2a9b01344.png"},{"id":84726598,"identity":"7a458a5f-ec69-437a-901d-c1af3ef13710","added_by":"auto","created_at":"2025-06-16 16:07:23","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2554156,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5557820/v1/9d2549f6-b4c8-4d07-81b4-f2d492b845d6.pdf"},{"id":81243255,"identity":"f7dc41a1-c974-4c52-8017-f9aecdc9946d","added_by":"auto","created_at":"2025-04-24 00:25:34","extension":"docx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":88225,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-5557820/v1/13b5a9e1b50eb1da16b1cd6a.docx"},{"id":81243085,"identity":"82a6ec04-c1ca-42e4-8cdd-cdfcf3d1864d","added_by":"auto","created_at":"2025-04-24 00:17:37","extension":"xls","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":152258785,"visible":true,"origin":"","legend":"","description":"","filename":"evendepth16s.xls","url":"https://assets-eu.researchsquare.com/files/rs-5557820/v1/5a29e31901b4d63506df861b.xls"},{"id":81243261,"identity":"22930cc2-cc14-4515-92f7-d66ea1fd0b02","added_by":"auto","created_at":"2025-04-24 00:25:34","extension":"xls","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":14957981,"visible":true,"origin":"","legend":"","description":"","filename":"evendepthits.xls","url":"https://assets-eu.researchsquare.com/files/rs-5557820/v1/411123523444797b9e1476da.xls"},{"id":81243087,"identity":"8f870177-5789-4a7d-bb7e-b3fe7d21a04d","added_by":"auto","created_at":"2025-04-24 00:17:40","extension":"xls","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":234284595,"visible":true,"origin":"","legend":"","description":"","filename":"filtered16s.xls","url":"https://assets-eu.researchsquare.com/files/rs-5557820/v1/c1461eac4e92404d86aac294.xls"},{"id":81243074,"identity":"399bd4d9-e286-4d0b-a86f-45722fe16715","added_by":"auto","created_at":"2025-04-24 00:17:34","extension":"xls","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":15260581,"visible":true,"origin":"","legend":"","description":"","filename":"filteredits.xls","url":"https://assets-eu.researchsquare.com/files/rs-5557820/v1/40787971b7f0be9e84befb8c.xls"}],"financialInterests":"","formattedTitle":"Effect of plant elicitor Isotianil on the rhizosphere soil microbiome composition of banana cultivars with different resistance to Fusarium wilt","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSoil-borne fungal diseases lead to global annual losses to crop production estimated at 15% (Newbery et al. 2016). Common control measures primarily include breeding resistant varieties, spatial and temporal diversification in planting, chemical control, and biological control (Noble et al. 2005). Although large-scale application of chemical pesticides or fungicides can be highly effective in the short term, the environmental and economic costs are substantial. Growing pathogen resistance to chemical control has been well documented. Consequently, extensive research continues to explore efficient and environmentally friendly control strategies to mitigate biotic stress damage and reduce risks to human health (Ruijven et al. 2020). Plant elicitors as resistance inducers are one option to reduce damage from fungal diseases (Zhou et al. 2023).\u003c/p\u003e\n\u003cp\u003eAs small molecules that trigger plant immune responses, elicitors can mimic natural elicitors or defense signaling molecules. They activate defense mechanisms by interacting with their homologous plant receptors or interfering with other defense signaling components (Thakur and Sohal 2013). Synthetic inducers have been successfully applied in crop protection, offering an attractive alternative to traditional biocidal agrochemicals (Bektas and Eulgem 2015). Isotianil, developed by Bayer, is a thiazole-based compound that lacks direct antibacterial or antifungal activity. However, it can activate defense responses against pathogens in different plant species (Ogawa et al. 2011). It has been utilized as a crop protection agent against rice blast and bacterial leaf blight in rice and wheat (Portz et al. 2021). Isotianil induces the accumulation of defense-related genes associated with salicylic acid signaling (e.g., \u003cem\u003eOsWRKY45\u003c/em\u003e) and defense-related enzymes (e.g., lipoxygenase or PAL) (Braun et al. 2014). Isotianil significantly induces resistance against banana sigatoka leaf spot and lasting up to 28 days (Qi et al. 2019). Moreover, synthetic inducers serve as effective tools in basic research methods, expanding our understanding of plant immunity.\u003c/p\u003e\n\u003cp\u003eThe rhizosphere microbiome is crucial for maintaining plant health and productivity (Berendsen et al. 2012; Qu et al. 2020). Plants can protect themselves from different types of stress by assembling a beneficial microbiome through various mechanisms (Trivedi et al. 2020). In response to pathogen infection, plants not only trigger systemic immune responses to directly inhibit pathogen invasion but also modulate the microbiome (Wei et al. 2020). This regulation includes altering the composition and quantity of root exudates, volatiles, and reactive oxygen species, thereby modifying the soil microbiome to directly or indirectly suppress disease occurrence (Wen et al. 2023). The microbial elicitor BAR11 enhances \u003cem\u003eArabidopsis thaliana\u003c/em\u003e resistance to \u003cem\u003ePseudomonas syringae\u003c/em\u003e pv. \u003cem\u003etomato\u003c/em\u003e DC3000 by influencing the accumulation of specific secondary metabolites and altering the composition and diversity of the rhizosphere microbial community, particularly promoting the recruitment of beneficial microorganisms that contribute to enhanced plant defense (Wang et al. 2024). Synthetic elicitors such as methyl salicylic acid and acibenzolar-s-methyl stimulate pine wilt resistance in plants and modulate the root microbiome, increasing the abundance of beneficial microorganisms (Mannaa et al. 2020).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBananas are the world\u0026apos;s most traded fruit and the fourth most important food crop, playing a vital role in ensuring the stability and progress of developing countries (Nayar 2010). However, banana production is seriously threatened by Fusarium wilt, caused by\u003cem\u003e\u0026nbsp;Fusarium oxysporum\u003c/em\u003e f. sp. \u003cem\u003ecubense\u003c/em\u003e (\u003cem\u003eFoc\u003c/em\u003e), which endangers the sustainable development of the banana industry (Siamak and Zheng 2018). While breeding for disease-resistant varieties has proven to be the most effective control measure, no commercial cultivars with complete immunity to the widely spread \u003cem\u003eFoc\u0026nbsp;\u003c/em\u003eTropic Race\u003cem\u003e\u0026nbsp;\u003c/em\u003e4 (TR4) have been developed (Ploetz 2015). Current control strategies primarily rely on strict quarantine measures, the cultivation of disease-tolerant varieties, and biological control. Traditional fungicides are not feasible because of their high cost and low efficacy. Biological control serves as an advantageous alternative (Pegg et al. 2019). However, the effectiveness of biocontrol agents is often inconsistent (Bardin et al. 2015),\u0026nbsp;especially for soil-borne diseases (Yang et al. 2019; Ptaszek et al. 2023). Therefore, understanding the mechanisms by which bananas recruit beneficial microbes related to disease resistance is crucial for managing Fusarium wilt (Dita et al. 2018). One promising research direction involves enhancing banana resistance through plant immunity elicitors, which may lead to new strategies for recruiting beneficial microbes. This approach not only aids in understanding the complex interactions between bananas and microorganisms but also provides a scientific basis and practical potential for disease management.\u003c/p\u003e\n\u003cp\u003eOur group demonstrated that the application of Isotianil could enhance the expression of genes related to plant resistance induction and starch synthesis in banana corms, indicating its potential to boost the host\u0026rsquo;s resistance to TR4 (Zhou et al. 2023). However, just how Isotianil impacts the belowground rhizosphere microbiome compositions remain unclear. Considering the significant role of the rhizosphere microbiome in the occurrence and development of Fusarium wilt, this study employed amplicon sequencing to analyze the effects of Isotianil-induced plant resistance on the rhizosphere microbiome of the susceptible banana cultivar Baxijiao and the resistant cultivar Yunjiao No. 1.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003eVarieties and cultivation methods\u003c/p\u003e\n\u003cp\u003eThe experiment was conducted using the Fusariam wilt-susceptible Cavendish banana cultivar Baxijiao and the tolerant Cavendish cultivar Yunjiao No. 1. Tissue-cultured plantlets were initially cultivated at 25 \u0026deg;C with a 16 h light / 8 h dark photoperiod for 20 days. After developing 4-5 leaves and establishing roots, the plantlets were transplanted into 5 cm trays filled with a mixture of coir and plantlet substrate (1:1). After 56 days, when approximately 3-4 new leaves had grown, the plantlets were transplanted into 25 cm diameter plastic pots filled with native horticultural red ferrosols soil which has not previously been exposed to bananas and unsterilized banana plantlet substrate (1:1).\u003c/p\u003e\n\u003cp\u003ePathogen and Isotianil inoculation disease index assessment\u003c/p\u003e\n\u003cp\u003eTR4 is the laboratory-maintained strain 15-1 (Zhang et al. 2018). After being cultured on Potato Dextrose Agar medium at 28 \u0026deg;C for 7 days, spores were collected by washing the plates with sterile water. The spore suspension concentration was determined to be 1\u0026times;10⁷ spores/mL using a hemocytometer. Isotianil suspension\u0026nbsp;(0.2 g/mL)\u0026nbsp;provided by Bayer Crop Science was applied at the time of transplantation into plastic pots, when the plants had approximately 8 leaves. Each banana plant received a root drench or foliar spray of 100 mL (0.07 mg/mL) Isotianil, applied every 28 days, for a total of three applications. 7 days after the second application of Isotianil, each plant was inoculated with 100 mL of the TR4 spore suspension (1\u0026times;10⁷ spores/mL) by wounding the roots, while the control treatment was treated with tap water. There were four treatments \u0026mdash; CK: control treatment; TA: inoculate TR4 alone; FT: foliar application and inoculate TR4; RT: root drenching and inoculate TR4 \u0026mdash; with 45 plants per treatment.\u0026nbsp;Wilt severity\u0026nbsp;was recorded after 62 further days similar as in our previous study (Zhang et al. 2019).\u0026nbsp;Wilt severity\u0026nbsp;was divided into four levels: 0 = healthy plant, 1 = 1 % ‒25 % necrosis in the corm, 2 = 26 % ‒ 50 %\u0026nbsp;necrosis in the corm, 3 = 51% ‒75%\u0026nbsp;necrosis in the corm, 4 = 75 % ‒100 %\u0026nbsp;necrosis in the corm.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eDisease Index = \u0026sum; (Number of Plants in Each\u0026nbsp;\u003c/em\u003e\u003cem\u003eWilt Severity\u003c/em\u003e\u003cem\u003e\u0026nbsp;\u0026times; Representative Value) / (Total Number of Plants \u0026times; 4) \u0026times; 100\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eSoil sample collection, DNA extraction, and PCR\u003c/p\u003e\n\u003cp\u003eBanana plants were removed from the soil and vigorously shaken to remove soil around the roots. \u0026nbsp; Each replicate consisted of 3 seedlings, resulting in 15 samples per treatment (A total of 120 samples). The soil adhering to the root surface was then collected using a brush and stored at -80 \u0026deg;C until DNA extraction. Total genomic DNA samples were extracted using the OMEGA Soil DNA Kit (D5625-01) from Omega Bio-Tek, Norcross, GA, USA, according to the protocol provided by the manufacturer. The extracted DNA samples were stored at -20 \u0026deg;C until further analysis. The concentration and purity of the DNA were assessed using a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA), while the integrity was checked via agarose gel electrophoresis. The V3\u0026ndash;V4 region of bacterial 16S rRNA genes was amplified using PCR with the forward primer 338F (5\u0026apos;-ACTCCTACGGGAGGCAGCA-3\u0026apos;) and the reverse primer 806R (5\u0026apos;-GGACTACHVGGGTWTCTAAT-3\u0026apos;). For fungal community analysis, the ITS1 region of fungal ITS rRNA genes was amplified using the forward primer ITS5F (5\u0026apos;-GGAAGTAAAAGTCGTAACAAGG-3\u0026apos;) and the reverse primer ITS1R (5\u0026apos;-GCTGCGTTCTTCATCGATGC-3\u0026apos;). The PCR system and program were referenced from our previous work (Zhang et al. 2024).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSequencing analysis\u003c/p\u003e\n\u003cp\u003eAfter individual quantification, the amplicons were pooled in equal proportions and sequenced using paired-end 2 \u0026times; 300 bp reads on an Illumina MiSeq platform, utilizing the MiSeq Reagent Kit v3, at Shanghai Personal Biotechnology Co., Ltd, Shanghai, China.\u0026nbsp;The raw reads were deposited in the NCBI Sequence Read Archive (16S: PRJNA949124; ITS: PRJNA949129). Quality-filtered and merged sequences were denoised using the DADA2 plugin (Callahan et al. 2016) in QIIME2 (Bolyen et al. 2019) to obtain amplicon sequence variants (ASVs). Chloroplast and mitochondrial sequences were removed from all samples. Taxonomic classification of ASVs was performed using the Naive Bayes classifier in QIIME2, based on the SILVA (v132) and UNITE (v8.0) database. To ensure comparability across samples, the sequencing depth was rarefied to 95 % of the minimum sequencing depth (16S: 36525; ITS:65063).\u0026nbsp;The ASV table is provided in \u003cstrong\u003eElectronic supplementary material 1\u003c/strong\u003e\u003cstrong\u003e\u0026ndash;\u003c/strong\u003e\u003cstrong\u003e4\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eData analysis\u003c/p\u003e\n\u003cp\u003eR version 4.1.2 was used for data analysis, with visualization performed using GraphPad Prism 10. Alpha diversity indices of the community were calculated using the vegan package. The vegan package was also employed to calculate differences in community composition between samples using Permutational Multivariate Analysis of Variance (PERMANOVA)\u0026nbsp;and Principal Co-ordinates Analysis (PCoA). Correlation analysis was performed using Spearman correlation. Differences in data between groups were analyzed using the Kruskal-Wallis test, followed by multiple comparison correction using the Benjamini-Hochberg (BH) method in SPSS 25.\u0026nbsp;A cross-kingdom co-occurrence network was constructed using the Hmisc package (R \u0026gt; 0.6,\u0026nbsp;adjusted\u0026nbsp;\u003cem\u003eP\u0026nbsp;\u003c/em\u003e\u0026lt; 0.05), with Spearman correlation and BH multiple testing corrections applied. The genus-level networks and indices were visualized and calculated using Gephi 10. A high modularity index and higher proportion of negative correlation edges indicate more stable community network, while a higher average degree represents more complex community network (Gao et al. 2021). The iCAMP, picante and ape package were used to construct phylogenetic-based null model analysis and calculate Beta nearest taxon indice (Beta NTI), the normalized stochasticity ratio (NST) was calculated with the NST package. To ensure the reliability of the randomization process, we performed 1000 randomizations for both Beta NTI and NST. The absolute value of Beta NTI lower than 2 indicates that random processes dominate during community assembly. A higher NST indicates a greater impact of stochasticity processes on community assembly (Ning et al. 2020). Fungal ASVs were assigned into functional guilds using the FUNGuild (Nguyen et al. 2016).\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eIncidence of Fusarium wilt in different varieties following TR4 and Isotianil inoculation\u003c/p\u003e\n\u003cp\u003eWhen inoculated with TR4 alone (TA) the disease index of Fusarium wilt in Yunjiao No. 1 (Disease index: 26.49) was significantly lower than in Baxijiao (51.56). The application of Isotianil (FT: foliar application and inoculate TR4; RT: root drenching and inoculate TR4) significantly reduced the disease index of Fusarium wilt in both Baxijiao (26.61in FT, 25.52 in RT) and Yunjiao No. 1 (11.98 in FT, 11.98 in RT) compared to inoculation with TA. There were no significant differences between RT and FT \u003cstrong\u003e(Fig. 1)\u003c/strong\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e[Insert Fig. 1]\u003c/p\u003e\n\u003cp\u003eBacterial and fungal communities\u0026rsquo; diversity and composition differences following TR4 and Isotianil inoculation\u003c/p\u003e\n\u003cp\u003eCompared to the control treatment (CK), the TA treatment showed no significant differences in the Chao1 and Shannon indices of bacterial and fungal communities in\u0026nbsp;both Baxijiao and Yunjiao No. 1. Compared to the TA treatment, FT and RT had no significant impact on the Chao1 and Shannon indices of bacterial and fungal communities in both Baxijiao\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eand Yunjiao No.1\u003cstrong\u003e\u0026nbsp;(Fig. 2)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e[Insert Fig. 2]\u003c/p\u003e\n\u003cp\u003eCompared to the CK, TA significantly changed the bacterial community composition in both Baxijiao and Yunjiao No. 1. Relative to TA, RT significantly changed the bacterial composition in both Baxijiao and Yunjiao No. 1, while FT only significantly affected the bacterial community composition in Yunjiao No. 1.\u0026nbsp;Compared to the CK, TA significantly changed the fungal community composition in Yunjiao No. 1. Compared to TA, both RT and FT significantly changed the fungal composition in Yunjiao No. 1, but had no significant impact on Baxijiao \u003cstrong\u003e(Fig. 3)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e[Insert Fig. 3]\u003c/p\u003e\n\u003cp\u003eResponses of bacterial and fungal taxa following TR4 and Isotianil inoculation\u003c/p\u003e\n\u003cp\u003eFor the dominant phylum (relative abundance \u0026gt; 1%), the relative abundance of Firmicutes significantly decreased compared to CK following TA in Baxijiao. Compared to TA, FT significantly decreased the relative abundance of Firmicutes in Baxijiao. RT increased the relative abundance of Ascomycota and decreased the relative abundance of Actinobacteria, Gemmatimonadetes and Rozellomycota in Baxijiao. Compared to CK, TA significantly decreased the relative abundance of Chloroflexi in Yunjiao No. 1. Compared to TA, FT significantly decreased the relative abundance of Firmicutes in Yunjiao No. 1. RT increased the relative abundance of Proteobacteria and Bacteroidetes, while decreased the relative abundance of Chloroflexi, Gemmatimonadetes and Mortierellomycota in Yunjiao No. 1 \u003cstrong\u003e(Fig. 4)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e[Insert Fig. 4]\u003c/p\u003e\n\u003cp\u003eFor dominant genera (relative abundance \u0026gt; 1%), compared to CK, TA increased the relative abundance of \u003cem\u003eFusarium\u003c/em\u003e in Baxijiao. Relative to TA, RT increased the relative abundance of \u003cem\u003eBlrii41\u003c/em\u003e, \u003cem\u003eDevosia\u003c/em\u003e, and \u003cem\u003eAcremonium\u003c/em\u003e, while decreased the relative abundance of \u003cem\u003eSphingomonas\u003c/em\u003e in Baxijiao. Relative to TA, FT significantly increased the relative abundance of \u003cem\u003eAcremonium\u003c/em\u003e in Yunjiao No. 1. RT increased the relative abundance of\u003cem\u003e\u0026nbsp;Blrii41\u003c/em\u003e, \u003cem\u003eDevosia\u003c/em\u003e, \u003cem\u003eAcremonium\u003c/em\u003e and \u003cem\u003ePlectosphaerella\u003c/em\u003e, while decreasing the relative abundance of\u003cem\u003e\u0026nbsp;KD4_96\u003c/em\u003e and \u003cem\u003eMND1\u003c/em\u003e in Yunjiao No. 1 \u003cstrong\u003e(Fig. 5)\u003c/strong\u003e. In addition, the \u003cem\u003eBlrii41\u003c/em\u003e, \u003cem\u003eDevosia\u003c/em\u003e, \u003cem\u003eAcremonium\u003c/em\u003e and \u003cem\u003ePlectosphaerella\u003c/em\u003e showed a significant negative correlation with disease index in Baxijiao and Yunjiao No. 1, across all TR4-inoculated samples\u003cstrong\u003e\u0026nbsp;(Table. 1)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e[Insert Fig. 5]\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1\u003c/strong\u003e Correlation between dominant bacterial and fungal genera and\u0026nbsp;Fusarium wilt disease index\u0026nbsp;based on Spearman across all TR4-inoculated samples.\u003c/p\u003e\n \u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"402\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 114px;\"\u003e\n \u003cp\u003egenus\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 144px;\"\u003e\n \u003cp\u003eBaxijiao\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 144px;\"\u003e\n \u003cp\u003eYunjiao No.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 78px;\"\u003e\n \u003cp\u003eR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 78px;\"\u003e\n \u003cp\u003eR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 114px;\"\u003e\n \u003cp\u003e\u003cem\u003eSubgroup_6\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 78px;\"\u003e\n \u003cp\u003e-0.015\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.705\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 78px;\"\u003e\n \u003cp\u003e0.058\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.924\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 114px;\"\u003e\n \u003cp\u003e\u003cem\u003eKD4-96\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 78px;\"\u003e\n \u003cp\u003e-0.050\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.011\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 78px;\"\u003e\n \u003cp\u003e0.374\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.743\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 114px;\"\u003e\n \u003cp\u003e\u003cem\u003eA4b\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 78px;\"\u003e\n \u003cp\u003e-0.316\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.943\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 78px;\"\u003e\n \u003cp\u003e0.011\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.034\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 114px;\"\u003e\n \u003cp\u003e\u003cem\u003eAD3\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 78px;\"\u003e\n \u003cp\u003e0.006\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.447\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 78px;\"\u003e\n \u003cp\u003e0.116\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.967\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 114px;\"\u003e\n \u003cp\u003e\u003cem\u003eRokubacteriales\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 78px;\"\u003e\n \u003cp\u003e0.103\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.537\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 78px;\"\u003e\n \u003cp\u003e0.094\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.502\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 114px;\"\u003e\n \u003cp\u003e\u003cem\u003eSBR1031\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 78px;\"\u003e\n \u003cp\u003e-0.197\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.144\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 78px;\"\u003e\n \u003cp\u003e0.221\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.195\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 114px;\"\u003e\n \u003cp\u003e\u003cem\u003eMND1\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 78px;\"\u003e\n \u003cp\u003e0.384\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.097\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 78px;\"\u003e\n \u003cp\u003e0.250\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.009\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 114px;\"\u003e\n \u003cp\u003e\u003cem\u003eSphingomonas\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 78px;\"\u003e\n \u003cp\u003e0.595\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.722\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 78px;\"\u003e\n \u003cp\u003e0.054\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.000\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 114px;\"\u003e\n \u003cp\u003e\u003cem\u003eBIrii41\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 78px;\"\u003e\n \u003cp\u003e-0.551\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.012\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 78px;\"\u003e\n \u003cp\u003e-0.370\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.000\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 114px;\"\u003e\n \u003cp\u003e\u003cem\u003eDevosia\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 78px;\"\u003e\n \u003cp\u003e-0.522\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.002\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 78px;\"\u003e\n \u003cp\u003e-0.446\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.000\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 114px;\"\u003e\n \u003cp\u003e\u003cem\u003eFusarium\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 78px;\"\u003e\n \u003cp\u003e-0.078\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.002\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 78px;\"\u003e\n \u003cp\u003e-0.446\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.613\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 114px;\"\u003e\n \u003cp\u003e\u003cem\u003eMortierella\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 78px;\"\u003e\n \u003cp\u003e0.057\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.619\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 78px;\"\u003e\n \u003cp\u003e0.076\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.712\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 114px;\"\u003e\n \u003cp\u003e\u003cem\u003eBotryotrichum\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 78px;\"\u003e\n \u003cp\u003e-0.381\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.602\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 78px;\"\u003e\n \u003cp\u003e-0.080\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.010\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 114px;\"\u003e\n \u003cp\u003e\u003cem\u003eGibberella\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 78px;\"\u003e\n \u003cp\u003e-0.015\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.003\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 78px;\"\u003e\n \u003cp\u003e0.428\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.924\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 114px;\"\u003e\n \u003cp\u003e\u003cem\u003eAcremonium\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 78px;\"\u003e\n \u003cp\u003e-0.495\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 78px;\"\u003e\n \u003cp\u003e-0.668\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 114px;\"\u003e\n \u003cp\u003e\u003cem\u003eDidymella\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 78px;\"\u003e\n \u003cp\u003e0.308\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.491\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 78px;\"\u003e\n \u003cp\u003e0.105\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.040\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 114px;\"\u003e\n \u003cp\u003e\u003cem\u003ePlectosphaerella\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 78px;\"\u003e\n \u003cp\u003e-0.300\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 78px;\"\u003e\n \u003cp\u003e-0.599\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.046\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\u003eEffects of TR4 and Isotianil inoculation on cross-kingdom co-occurrence networks\u003c/p\u003e\n\u003cp\u003eCompared to CK, TA resulted in decreased network modularity index, proportion of cross-kingdom edges, and average degree in Baxijiao \u003cstrong\u003e(Fig.\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e6\u003c/strong\u003e\u003cstrong\u003ea) (Table. 2)\u003c/strong\u003e. In Yunjiao No. 1, the modularity index and the proportion of negative correlation edges increased \u003cstrong\u003e(Fig.\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e6\u003c/strong\u003e\u003cstrong\u003eb)\u003c/strong\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eRelative to TA, both FT and RT increased the modularity index and proportion of negative correlation edges of the network in Baxijiao and Yunjiao No. 1 \u003cstrong\u003e(Fig.\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e6\u003c/strong\u003e\u003cstrong\u003ea and b)\u003c/strong\u003e, but there was no significant change in the average degree \u003cstrong\u003e(Fig.\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e6\u003c/strong\u003e\u003cstrong\u003ec and d)\u003c/strong\u003e. Additionally, compared to CK, TA reduced the proportion of cross-kingdom edges in Baxijiao while increasing it in Yunjiao No. 1, indicating a decrease in the interaction between soil bacteria and fungi in the rhizosphere of Baxijiao and an enhancement in Yunjiao No. 1 \u003cstrong\u003e(Table. 2)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e[Insert Fig. 6]\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2\u003c/strong\u003e The number of edges in cross-kingdom Co-occurrence Networks.\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"527\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003eGenotype\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 68px;\"\u003e\n \u003cp\u003eTreatment\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003ePFF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003eNFF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003ePBF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003eNBF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003ePBB\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003eNBB\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"4\" style=\"width: 66px;\"\u003e\n \u003cp\u003eBaxijiao\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 68px;\"\u003e\n \u003cp\u003eCK\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e404\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e101\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e116\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e517\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e367\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 68px;\"\u003e\n \u003cp\u003eTA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e538\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e95\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e323\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e259\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 68px;\"\u003e\n \u003cp\u003eFT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e66\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e186\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e146\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e469\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e259\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 68px;\"\u003e\n \u003cp\u003eRT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e220\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e242\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e220\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e335\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e266\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"4\" style=\"width: 66px;\"\u003e\n \u003cp\u003eYunjiao No.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 68px;\"\u003e\n \u003cp\u003eCK\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e446\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e38\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e66\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e70\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e406\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e257\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 68px;\"\u003e\n \u003cp\u003eTA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e161\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e151\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e124\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e414\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e232\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 68px;\"\u003e\n \u003cp\u003eFT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e161\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e59\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e222\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e207\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e389\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e315\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 68px;\"\u003e\n \u003cp\u003eRT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e112\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e134\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e132\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e319\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e270\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eNote: PFF: positive correlation edge between fungi and fungi; NFF: negative correlation edge between fungi and fungi. PBF: positive correlation edge between bacteria and fungi. NBF: negative correlation edge between bacteria and fungi. PBB: positive correlation edge between bacteria and bacteria. NBB: negative correlation edge between bacteria and bacteria.\u003c/p\u003e\n\u003cp\u003eAssembly processes of bacterial and\u0026nbsp;fungal\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003ecommunities following TR4 and Isotianil inoculation\u003c/p\u003e\n\u003cp\u003eIn the null model, stochasticity processes played a more important role than deterministic ones during the bacterial\u0026nbsp;and\u0026nbsp;fungal\u003cem\u003e\u0026nbsp;\u003c/em\u003ecommunity assembly under all treatments \u003cstrong\u003e(Fig.\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e7a and Fig. S1a\u003c/strong\u003e\u003cstrong\u003e)\u003c/strong\u003e. For bacterial community, compared to CK, TA had a higher NST index in Baxijiao, and lower in Yunjiao No.1. Compared to TA, FT and RT significantly reduced the impact of stochasticity processes in Baxijiao bacterial\u003cem\u003e\u0026nbsp;\u003c/em\u003ecommunity assembly, while it increased the impact of stochasticity processes in Yunjiao No.1 \u003cstrong\u003e(Fig.\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e7b\u003c/strong\u003e\u003cstrong\u003e)\u003c/strong\u003e.\u0026nbsp;However, in the NST analysis of the fungal community, the NST of the fungal community showed significant deviation, with some values exceeding the expected range of 0 to 1. This suggests that NST analysis may not be applicable to the fungal community in this study\u003cstrong\u003e\u0026nbsp;(Fig. S1b)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e[Insert Fig. 7]\u003c/p\u003e\n\u003cp\u003eFUNGuild functional prediction of fungal community\u003c/p\u003e\n\u003cp\u003eCompared to CK, TA treatment did not result in significant differences in predictions of functional fungal guilds both in Baxijiao and Yunjiao No.1. However, compared to TA, the relative abundance of the Pathogen-Saprotroph-Symbiotroph guild significantly increased in Baxijiao under RT and FT treatments, and significantly increased in Yunjiao No.1 under the RT treatment, the relative abundance of Symbiotroph\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eguild significantly\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003edecreased in Yunjiao No.1 under the RT treatment\u003cstrong\u003e\u0026nbsp;(Table. S1 and S2)\u003c/strong\u003e. For the plant pathogens within the functional group, no significant differences were observed among treatments \u003cstrong\u003e(Fig. S2)\u003c/strong\u003e.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eRegulating the soil microbiome to reduce the occurrence of soil-borne diseases has been widely applied in crop production (Gang et al. 2013; Bubici et al. 2019; Fan et al. 2023). With advances in high-throughput sequencing technology, the underlying mechanisms have been further explored and optimized (Zhu et al. 2023). In addition to reducing the abundance of pathogens, research has demonstrated how to increase the number of beneficial microorganisms in the soil through direct inoculation or other measures (Tao et al. 2020). However, in addition to the efficient interaction with target crops, the effectiveness of directly inoculated beneficial microorganisms relies on their ability to successfully colonize the soil. Microorganisms must adapt to the diverse soil environment and overcome the self-regulatory mechanisms of the native microbiome. Consequently, the effectiveness of many high-efficiency microorganisms often remains inconsistent across different field conditions (Chen et al. 2022). By studying the beneficial microbial communities recruited in different resistant varieties, we can better develop effective disease control strategies. Microorganisms actively recruited by plants tend to be more competitive in the soil, enhancing the plant\u0026apos;s immune system, promoting healthy growth, and helping to protect against pathogen invasion (Rolfe et al. 2019).\u003c/p\u003e\n\u003cp\u003eIsotianil application enhances the expression of genes related to plant resistance induction and starch synthesis (Zhou et al. 2023). Research on elicitor-induced host resistance has primarily focused on the expression of the plant\u0026apos;s own resistance genes (Benhamou 1996; Zhai et al. 2017). However, the soil microbiome has proven to be closely related to disease development. Investigating the soil microbiome will enhance our understanding of disease progression (Wei et al. 2019). In this study, root drenching had a more pronounced impact on soil microorganisms compared to foliar application, likely due to the direct disturbance caused by Isotianil as a carbon source. Foliar application of Isotianil had no significant effect on the bacterial and fungal community composition in Baxijiao. For Yunjiao No.1, on the other hand, both foliar and root applications of Isotianil significantly altered the bacterial and fungal communities. \u0026nbsp;The number of changes in the bacterial and fungal phyla and genera of Yunjiao No.1 was greater than that of Baxijiao. This may be due to more active root exudates or higher rhizosphere metabolic activity in Yunjiao No.1, which consequently altered the abundance of more microbial taxa. Subsequent research could involve analyzing the composition of root exudates after foliar application to link microbial community taxa with the exudates, further confirming the reasons behind this stronger variation. Once these microbes colonize the rhizosphere, they can enhance plant resistance through various mechanisms such as inhibiting pathogens, promoting plant growth, and inducing plant resistance (Kwak et al. 2018; Mendes et al. 2018b). For example, Liu (2023) found that banana varieties\u0026apos; resistance to the TR4 is linked to their ability to recruit \u003cem\u003eTrichoderma\u003c/em\u003e and \u003cem\u003ePenicillium\u003c/em\u003e, which are stimulated by D-(-)-ribofuranose and propylene glycol. Enhancing soils with these exudates improved resistance in susceptible varieties.\u003c/p\u003e\n\u003cp\u003eCompared to inoculation with TR4 alone, root drenching Isotianil significantly increased the relative abundances of \u003cem\u003eBlrii41\u003c/em\u003e, \u003cem\u003eDevosia\u003c/em\u003e, and \u003cem\u003eAcremonium\u003c/em\u003e in the rhizosphere of both banana varieties. These microorganisms are all potentially beneficial. \u003cem\u003eBlrii41\u003c/em\u003e may be associated with nutrient transformation and is typically abundant in organic soils (Stephenson and Kahlert 2018; Wei et al. 2024). \u003cem\u003eDevosia\u003c/em\u003e and \u003cem\u003eAcremonium\u003c/em\u003e have been shown to play important roles in microbe-mediated disease resistance (Grunewaldt-St\u0026ouml;cker and von Alten 2003; Barros et al. 2022). For example, the non-pathogenic strain \u003cem\u003ePseudomonas syringae\u003c/em\u003e DC3000 can reduce the secretion of myristic acid by tomato roots, leading to an increase in the abundance of \u003cem\u003eDevosia\u003c/em\u003e in the rhizosphere, which ultimately promotes plant growth and induces the formation of soil-borne disease suppression (Liu et al. 2024). The endophytic fungus \u003cem\u003eAcremonium\u003c/em\u003e isolated from \u003cem\u003eLium davidii\u003c/em\u003e has been shown to effectively inhibit various pathogenic fungi and promote plant growth (Khan et al. 2021). Therefore, after the application of Isotianil, the microorganisms recruited in the banana rhizosphere exhibit disease-suppressive potential, which may enhance their ability to resist pathogens.\u003c/p\u003e\n\u003cp\u003eA higher modularity index indicates greater community stability, which is a mark of healthy soil and the recruitment of beneficial microorganisms (Gao et al. 2021; Hassani et al. 2023). The microbial community network in soils resistant to banana wilt had a higher proportion of negative correlations, which may lead to greater stress due to pathogen infection (Lv et al. 2023). In this study, compared to CK, the stability of the microbial community\u0026nbsp;network in Baxijiao decreased after TR4 inoculation, while in Yunjiao No.1, the stability increased. This reflects Yunjiao No.1\u0026apos;s stronger microbial regulatory capacity after TR4 infection, shaping more stable network interactions. This phenomenon is also observed in other species. For instance, tobacco varieties resistant to black shank disease (\u003cem\u003ePhytophthora parasitica\u003c/em\u003e) have more complex and highly connected microbial networks than susceptible varieties (Li et al. 2023). Similarly, common bean varieties resistant to Fusarium wilt possess more complex communities than susceptible varieties (Mendes et al. 2018a). The application of Isotianil improved the stability of the rhizosphere microbiome\u0026nbsp;network, likely by inducing systemic resistance in plants, which enhanced the recruitment of beneficial microorganisms. This elicitor effect is similar to that of biocontrol microorganisms in inducing plant resistance (Cardoni et al. 2023; Ge et al. 2024). Yunjiao No.1 exhibited a stronger response to both TR4 and Isotianil than Baxijiao, and the combination of Yunjiao No.1 with Isotianil demonstrated significant disease resistance potential, which may provide an effective disease control strategy for the banana industry.\u003c/p\u003e\n\u003cp\u003eA higher stochastic process indicates lower environmental selection pressure (Du et al. 2021). After Isotianil application, Baxijiao exhibited a decrease in the Beta nearest taxon index in bacterial communities, while Yunjiao No.1 showed an increase in the Beta nearest taxon index. This suggests that the two banana varieties respond differently to Isotianil. In Baxijiao, the reduced randomness may imply that Isotianil application strengthens its selective control over rhizosphere microorganisms. As environmental selection pressure increases, the microbial community structure becomes more deterministic, which might reflect Baxijiao\u0026apos;s strategy to enhance disease resistance by reducing community randomness (Wen et al. 2022). In contrast, Yunjiao No.1, despite showing increased determinism process after TR4 inoculation, experienced greater community stochastic process after Isotianil application, possibly due to a reduced need for strict community assembly control as disease incidence decreased. These differences provide important insights for understanding and improving disease resistance in different banana varieties. Considering cultivar-specific rhizosphere microbial assembly patterns could be a key factor in disease resistance breeding and control strategies.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThrough this study, we gained insights into how inducers promote the recruitment of beneficial microorganisms by plants, providing a theoretical basis for optimizing soil microbial communities. However, further research is needed to verify the specific roles of these microbial species in maintaining banana health, especially under field conditions. Additionally, metabolomic analysis to identify differences in root exudates could better reveal the mechanisms behind microbial recruitment, helping to identify which beneficial microorganisms have stronger interactions with bananas. By customizing the application of these microbial agents and optimizing soil microbial communities, we can further enhance banana disease resistance. Taking these factors into account, more precise and effective plant disease resistance strategies can be developed, improving crop health and yield, reducing pesticide use, and advancing sustainable agriculture.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThe application of Isotianil not only enhances plant disease resistance but also regulates the stability of the rhizosphere microbiome, promoting the recruitment of beneficial microorganisms. The disease-resistant cultivar Yunjiao No. 1, when combined with Isotianil, demonstrated significant disease resistance effects and beneficial microbiome regulation. These findings provide novel scientific evidence and potential applications for enhancing the disease resistance of banana varieties and improving agricultural practices.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by National Natural Science Foundation of China (NSFC32161143001); Science and Technology Department of Yunnan Provincial Government (202204BI090019), Yunnan Fundamental Research Project (grant No. 202401AT070094) and Yunnan \u0026nbsp;Fundamental \u0026nbsp; Research \u0026nbsp;Project (grant No. 202401BD070001-096) and Yunling Scholar Programme of Yunnan Provincial Government (YNWR-YLXZ-2018-018); Funded in part by Seedqual Initiative of One CGIAR, and the International Atomic Energy Agency, under the project \u0026ldquo;An Integrative Approach to Enhancing Disease Resistance Against Fusarium Wilt (\u003cem\u003eFoc\u003c/em\u003e TR4) in Banana \u0026ndash;Phase II (D23033).\u0026rdquo; We thank Glenn Hyman from of the Alliance of Bioversity International and CIAT Science Writing Service for his editorial support of this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWenlong Zhang: Methodology, Software, Visualization, Writing - review \u0026amp; editing. Guangdong Zhou: Methodology, Visualization. Ping He: Visualization, Supervision, Investigation. Hongwei Yu: Visualization, Software, Investigation. Libo Tian: Visualization, Supervision, Investigation. Yun-Yue Wang: Conceptualization, Supervision. Si-Jun Zheng: Conceptualization, Supervision, Funding acquisition, Writing - review \u0026amp; editing. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eResearch data have been deposited in the NCBI databases (16S: PRJNA 949124; ITS: PRJNA949129).\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eBardin M, Ajouz S, Comby M, Lopez-Ferber M, Graillot B, Siegwart M, Nicot PC (2015). Is the efficacy of biological control against plant diseases likely to be more durable than that of chemical pesticides? 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Plant Soil 1-15. https://doi.org/10.1007/s11104-024-06709-4\u003c/li\u003e\n \u003cli\u003eZhou GD, He P, Tian L, Xu S, Yang B, Liu L, Wang Y, Bai T, Li X, Li S, Zheng SJ (2023) Disentangling the resistant mechanism of Fusarium wilt TR4 interactions with different cultivars and its elicitor application. Front Plant Sci 14:1145837. https://doi.org/10.3389/fpls.2023.1145837\u003c/li\u003e\n \u003cli\u003eZhu Z, Wu G, Deng R, Hu X, Tan H, Chen Y, Tian Z, Li J (2023) Spatiotemporal biocontrol and rhizosphere microbiome analysis of Fusarium wilt of banana. Commun Biol 6:27. https://doi.org/10.1038/s42003-023-04417-w\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"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":"Plant health, Plant elicitor, Rhizosphere, 16S and ITS, Co-occurrence network, Bacterial community assembly","lastPublishedDoi":"10.21203/rs.3.rs-5557820/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5557820/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eAims\u003c/h2\u003e \u003cp\u003eBanana Fusarium wilt disease severely threatens the sustainable development of the banana industry. Regulating the local microbiome to reduce disease incidence offers broader adaptability, with a potential exploration into plant-induced recruitment of beneficial microbes. Our previous study showed that the plant elicitor Isotianil induces resistance to banana Fusarium wilt. This study assessed the impact of Isotianil on soil microbiomes.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003ePot experiments were conducted with the susceptible banana cultivar Baxijiao and the tolerant cultivar Yunjiao No. 1, inoculating them with the Fusarium pathogen and introducing Isotianil by foliar application and root drenching. Amplicon sequencing was used to analyze the rhizosphere bacterial and fungal communities.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eResults showed that Isotianil had 48% and 55% disease control efficacy rates for Baxijiao and Yunjiao No. 1 after foliar application, and 51% and 55% after root drenching, respectively. Amplicon sequencing revealed that, compared to pathogen alone, foliar application of Isotianil significantly altered the bacterial and fungal community compositions in Yunjiao No. 1, while root drenching significantly changed these communities in both cultivars. Root drenching of Isotianil notably increased the relative abundance of potential beneficial microbes such as \u003cem\u003eBlrii41\u003c/em\u003e, \u003cem\u003eDevosia\u003c/em\u003e, and \u003cem\u003eAcremonium\u003c/em\u003e and enhanced the stability of bacterial and fungal cross-kingdom networks. Additionally, the application of Isotianil increases the impact of stochastic processes in the bacterial community assembly of Baxijiao while decreasing their importance in Yunjiao No.1.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eThese findings suggest that Isotianil application modulates the rhizosphere microbiome, promoting beneficial microbes, stabilizing microbial networks, potentially contributing to disease suppression.\u003c/p\u003e","manuscriptTitle":"Effect of plant elicitor Isotianil on the rhizosphere soil microbiome composition of banana cultivars with different resistance to Fusarium wilt","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-24 00:17:28","doi":"10.21203/rs.3.rs-5557820/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2025-04-22T02:54:01+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-04-22T02:51:12+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-04-20T09:22:31+00:00","index":"","fulltext":""},{"type":"submitted","content":"Plant and Soil","date":"2025-04-17T05:36:18+00:00","index":"","fulltext":""},{"type":"decision","content":"Minor revisions","date":"2025-01-22T01:31:14+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":"ef9f5002-3cfe-4b56-9474-fbb94eca2fa2","owner":[],"postedDate":"April 24th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-06-16T16:04:02+00:00","versionOfRecord":{"articleIdentity":"rs-5557820","link":"https://doi.org/10.1007/s11104-025-07569-2","journal":{"identity":"plant-and-soil","isVorOnly":false,"title":"Plant and Soil"},"publishedOn":"2025-06-13 15:57:06","publishedOnDateReadable":"June 13th, 2025"},"versionCreatedAt":"2025-04-24 00:17:28","video":"","vorDoi":"10.1007/s11104-025-07569-2","vorDoiUrl":"https://doi.org/10.1007/s11104-025-07569-2","workflowStages":[]},"version":"v1","identity":"rs-5557820","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5557820","identity":"rs-5557820","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}