Foliar Application of Bacillus thuringiensis Enhances Tea Quality and Plant Defense via Phyllosphere Microbiome Modulation

Abstract

The plant microbiome plays a crucial role in the health of the tea plant, while Bacillus thuringiensis (Bt) is widely utilized as a biological pesticide in tea gardens, promoting sustainable agricultural practices. However, the effects of Bt spraying on tea quality and the structure and function of the phyllosphere microbiome remain unclear. This study evaluated the effects of Bt spraying on tea quality, microbiome composition, diversity, and potential functions using tea leaf quality measurements and high-throughput sequencing of the 16S/ITS rDNA genes. Results showed that spraying Bt1 significantly increased the contents of free amino acids (by 15.27%), flavonoids (by 18.00%), soluble sugars (by 62.55%), and key compounds such as epicatechin gallate (by 10.50%), gallocatechin gallate (by 122.52%), and epigallocatechin gallate (by 61.29%), leading to improved leaf quality. Co-occurrence network analysis indicated that the community structure of both epiphytic and endophytic microbes became more complex after Bt treatment. The abundance of beneficial bacteria, such as Novosphingobium, Methylobacterium, and Sphingomonas, increased significantly, while pathogenic fungi like Aspergillus and Phyllosticta decreased. Functional prediction indicated enhanced amino acid metabolism, secondary metabolism, and carbohydrate metabolism, particularly the biosynthesis of flavonoids, which supports disease resistance and boosts secondary metabolite levels. Furthermore, Bt application reduced pathogenic fungi, enhancing the tea plant’s resistance to diseases. Overall, foliar spraying of Bt can positively alter the phyllosphere microbiome by enriching beneficial bacteria and improving metabolic functions, ultimately enhancing tea plant resistance and quality, and providing a scientific basis for sustainable pest management in tea cultivation.

1. Introduction

The tea plant, scientifically known as Camellia sinensis (L.) Kuntze, is one of the most economically significant perennial crops globally, serving as the foundation of a multibillion-dollar industry that supports the livelihoods of millions of farmers, particularly in Asia [1]. China, the world’s largest tea producer, contributes over 40% of global tea production, with tea leaves not only being a culturally cherished beverage but also a critical agricultural commodity for international trade [2]. However, tea cultivation faces persistent challenges from pests and diseases, including tea geometrids (Ectropis grisescens), tea green leafhoppers (Empoasca onukii), and fungal pathogens like anthracnose (Colletotrichum spp.), which collectively cause annual yield losses of 10–30% and severely compromise leaf quality [3].

To mitigate these threats, chemical pesticides have long been the primary method of control for tea farmers [4]. Yet, their overuse has led to alarming issues such as the emergence of resistant pest populations, pesticide residues in tea products, and environmental contamination, posing risks to human health, ecosystem stability, and sustainable tea production [5,6,7]. In this context, microbial-based agents such as Bacillus thuringiensis (Bt) have emerged as sustainable and targeted alternatives for pest management in tea cultivation [8]. Bt, a well-characterized entomopathogen, produces insecticidal crystal proteins (Cry toxins) that selectively target pests like lepidopteran larvae (e.g., E. grisescens) and hemipteran insects (e.g., E. onukii). Field studies in China have demonstrated a reduction in pest population by 60–85% and significant mitigation of yield loss [9,10]. Specific Bt strains, such as B. thuringiensis var. Kurstaki, further disrupt pest life cycles through ovicidal and larvicidal activities, offering a reduced reliance on synthetic chemicals [11]. Beyond direct pest control, emerging evidence suggests that Bt applications may indirectly enhance plant health by modulating phyllosphere microbial communities or stimulating systemic resistance [9]; however, such mechanisms remain underexplored in tea systems.

The phyllosphere, a critical microbial habitat on plant surfaces, hosts diverse epiphytic and endophytic communities that contribute to host defense through antimicrobial synthesis, nutrient competition, and immune priming [12,13]. However, synthetic pesticides such as imidacloprid disrupt these protective microbiomes [14]. For instance, neonicotinoid applications reduce bacterial diversity in crops like tobacco (Nicotiana tabacum) and wheat (Triticum aestivum), depleting beneficial taxa like Proteobacteria [15,16], while long-term pesticide use destabilizes soil microbial networks [14]. In contrast, microbial biocontrol agents like Bt may offer a promising alternative. Bacillus spp. are known to enhance disease resistance in crops such as tomatoes and wheat by promoting growth, suppressing pathogens via antifungal metabolites, and stimulating systemic immunity [9,17,18]. Previous studies have also demonstrated that Bacillus thuringiensis can promote the growth of crops such as chickpea and spinach by inhibiting pathogenic microorganisms [19,20]. Despite these cross-crop insights, the specific effects of Bt on tea phyllosphere communities remain largely unexplored. Key questions persist: Does Bt restructure microbial diversity or functional guilds in tea leaves? Can it enrich beneficial taxa while suppressing pathogens? How do microbial shifts correlate with tea quality and plant resilience?

This study investigated the interplay between Bt application intensity, phyllosphere microbiome dynamics, and tea quality through controlled pot experiments. By integrating 16S/ITS rDNA sequencing with metabolomic profiling, we evaluated how Bt influenced microbial diversity and function while assessing biochemical indicators of tea quality and stress adaptation. Our research addressed three critical gaps: (1) Bt’s role as a microbial community modulator, (2) the correlation between microbial functional shifts and metabolite synthesis, and (3) the potential for Bt-induced microbiome restructuring to enhance systemic resistance. By positioning Bt as a dual-action agent that serves both as a pest control tool and as a microbiome engineer, this work establishes a scientific foundation for optimizing sustainable tea production strategies that harmonize ecological integrity with the achievement of high-quality yields.

2. Materials and Methods

2.1. Experimental Design

The experiment was conducted in a greenhouse at Wuyi University, Wuyishan City, Fujian Province, China (longitude: 118°00′35″ E, latitude: 27°44′25″ N) (Figure S1). One-year-old Camellia sinensis “Rougui” seedlings were first surface-sterilized with 6% H₂O₂ for 10 min and then transplanted into buckets containing 3 kg of soil each. The baseline soil fertility was measured as follows: pH 4.43, available nitrogen 28.21 mg/kg, available phosphorus 10.12 mg/kg, available potassium 168.26 mg/kg, and organic matter content 14.56 g/kg. Each bucket contained five tea seedlings, and the entire experiment was repeated three times, resulting in three biological replicates per treatment. A Bacillus thuringiensis (Bt) solution with a concentration of 32,000 IU/g was used, and the tea plant leaves were sprayed every 7 days. Three treatment groups were established: a control group with no spraying (CK), a group sprayed three times over a period of 21 days (Bt1), and a group sprayed six times over a period of 42 days (Bt2).

Initially, 150 mL of a 0.1% Hoagland nutrient solution was added to each pot. The pots were then placed in a greenhouse that maintained a temperature of 25 ± 2 °C and a relative humidity of 70 ± 5% during the experiment. To maintain appropriate nutrient levels, an additional 30 mL of the same concentration of nutrient solution was supplied to each pot every three days throughout the cultivation period, ensuring healthy plant growth.

All groups were sampled uniformly on day 43, with one bud and two leaves collected from each tea plant and then placed in sterile plastic bags. These samples were promptly transported to the laboratory and stored at −80 °C for subsequent analyses of phyllosphere microbial DNA, leaf enzyme activity, and leaf quality content.

2.2. Determination of Tea Quality-Related Components and Enzyme Activities

The quantification of tea polyphenols was performed using the Folin–Ciocalteu colorimetric method [21]. Caffeine analysis was conducted through high-performance liquid chromatography (HPLC, Thermo Fisher Scientific Co., Ltd., Waltham, MA, USA) (Figure S2) [22]. Free amino acid content was measured using the ninhydrin colorimetric method (UV-Vis, TU-1901, Beijing Puxia General Instrument Co., Ltd., Beijing, China) [23]. For the measurement of soluble sugar content, the anthrone method was employed [24]. Catechin compounds were quantitatively analyzed using the HPLC (Thermo Fisher Scientific Co., Ltd., Waltham, MA, USA) methodology (Figure S2) [25].

Enzymatic activities were assessed using established biochemical methods. Specifically, superoxide dismutase (SOD) activity was measured using the nitroblue tetrazolium reduction method [26], while peroxidase (POD) activity was measured using the guaiacol oxidation method [27]. Polyphenol oxidase (PPO) activity was quantified by monitoring the enzymatic oxidation rate of catechol substrate through spectrophotometric analysis (TU-1901, Beijing Puxia General Instrument Co., Ltd., Beijing, China) [28]. Finally, malondialdehyde (MDA) concentration, as an indicator of lipid peroxidation, was measured using the thiobarbituric acid reactive substance (TBAR) assay [29].

2.3. Collection of Phyllosphere Microorganisms from Tea Leaves

Epiphytic bacteria collection: Freshly harvested tea leaves were placed into a 250 mL Erlenmeyer flask containing 200 mL of sterile 0.01 mol/L phosphate-buffered saline (PBS) solution maintained at 4 °C. The mixture was subjected to ultrasonication for 15 min to facilitate the detachment of epiphytic microorganisms from the leaf surface. Subsequently, the flask was shaken at 200 rpm for 1 h at room temperature (20 °C) to enhance the extraction process. Following agitation, the resultant suspension was filtered through a sterile 0.22 μm cellulose membrane to capture the microorganisms, which were then sliced into 2 × 2 mm sections for further analysis [30].

Endophytic bacteria collection: The collected tea leaves underwent a rigorous surface disinfection process. Initially, the leaves were immersed in 75% ethanol for 1 min, followed by soaking in 1% sodium hypochlorite solution for 3 min and a final immersion in 75% ethanol for 30 s. The leaves were subsequently rinsed three times with sterile distilled water to ensure the complete removal of any contaminants. After disinfection, the leaves were subjected to freeze-drying and homogenization to prepare samples for endophytic bacterial extraction [30].

2.4. Extraction of Phyllosphere Microbial DNA from Tea Leaves

DNA extraction from the treated leaf samples (both epiphytic and endophytic) was conducted using the FastDNA^®Spin Kit for Soil (MP Biomedical, Solon, OH, USA) according to the manufacturer’s instructions. The integrity and purity of the extracted DNA were assessed through 1% agarose gel electrophoresis, while the concentration and purity were quantified using a NanoDropOne spectrophotometer. For PCR amplification and subsequent product analysis, genomic DNA served as the template. Based on the selected sequencing region, barcoded primers in conjunction with PremixTaq (TaKaRa) (TaKaRa Bio Inc., Kusatsu, Japan) were used for PCR amplification. The concentrations of PCR products were analyzed using GeneTools Analysis Software (Version 4.03.05.0, SynGene), and the necessary volume for each sample was determined based on the principle of equal mass, after which the PCR products were pooled together. The combined PCR products were purified using the E.Z.N.A.@Gel Extraction Kit (Omega Bio-tek, Inc., Norcross, GA, USA), and target DNA fragments were eluted in TE buffer. The DNA concentration of the eluted samples was subsequently measured using a NanoDrop 2000C Spectrophotometer (Thermo Scientific, Waltham, MA, USA). Only DNA samples meeting the quality standards were used for microbial community high-throughput sequencing analysis.

2.5. 16S/ITS rDNA High-Throughput Sequencing Analysis

Both 16S rDNA and ITS rDNA of the tea plant leaves were amplified to analyze the microbial community present. Detailed information regarding the primers and thermal cycling conditions is provided in Table S1. The PCR was performed using an ABI GeneAmp^® 9700 thermal cycler. Subsequently, the resulting amplicon library was sequenced using the PE250 sequencing method on the Illumina platform (Guangdong Magigene Biotechnology Co., Ltd., Guangzhou, China). The concentration of the amplicon library was measured with a Qubit 4.0 fluorometer (Thermo Fisher Scientific, Waltham, MA, USA). To ensure data quality, low-quality sequences, including those with an average quality score (Q < 20) and short lengths (<100 bp), were filtered out to yield the final effective dataset, termed “Effective Tags.” The Effective Tags from all samples were then clustered using Uparse, with a 97% sequence similarity threshold to define operational taxonomic units (OTUs). Subsequently, species annotation of the OTU sequences was performed using the BLAST (v2.12.0) method within Qiime framework, referencing the SILVA (v138) and the UNITE (v7.2) databases. This process allowed for the determination of microbial abundance at various taxonomic levels, providing insights into the composition of the microbial community associated with the tea plant leaves.

2.6. Data Analysis

Data conforming to normal distribution (as assessed by the Shapiro–Wilk test) were analyzed via one-way ANOVA with Duncan’s post hoc test (Kruskal–Wallis H, p < 0.05) using IBM SPSS (version 26, New York, NY, USA) software. Before performing α-diversity and β-diversity analyses based on the Bray–Curtis algorithm, the phyllosphere microbiome data were normalized. Diversity indices were subsequently visualized using GraphPad Prism (version 9.5, San Diego, CA, USA). A co-occurrence network was constructed using the “igraph” package in R software (version 4.3.1, Boston, TX, USA) based on Spearman correlations (r > 0.7, and p < 0.05), and these networks were visualized with Gephi (version 0.9.7, Paris, France) and Cytoscape (version 3.9.1, San Diego, CA, USA). Additionally, functional prediction for bacterial and fungal communities within the phyllosphere microbiome were performed using the PICRUST-KEGG and FUNGuild databases, respectively.

3. Result

3.1. The Effect of Bt Spraying on the Quality and Enzyme Activity of Tea Leaves

To evaluate the effect of Bt spraying on the quality of tea leaves, we analyzed 12 biochemical indicators (Figure 1A and Figure S2). Specifically, compared with the CK group, the Bt1 treatment significantly increased soluble sugar, flavonoids, and free amino acids (AAs) by 62.5%, 36.3%, and 18%, respectively (p < 0.05). Concurrently, caffeine was significantly reduced by 36.4% (p < 0.05). Under the Bt2 treatment, soluble sugar significantly increased by 36.2% (p < 0.05), while flavonoids and AAs showed slight increases of 7.3% and 2.2%, respectively. Moreover, caffeine significantly decreased by 17.8% (p < 0.05), and the tea polyphenol (TP) content decreased by 3.6%. These indicators, including TPs, AAs, caffeine, flavonoids, and soluble sugar, suggest that leaf spraying of Bt positively affects tea quality. Particularly under the Bt1 spraying intensity, the levels of flavonoids, soluble sugar, and AAs were significantly higher than those in the CK group (p < 0.05), and the treated leaves exhibited lower caffeine and TP contents compared to the CK group. Additionally, catechin analysis revealed that the contents of EGC, GC, ECG, GCG, and EGCG were significantly increased after Bt spraying compared to the CK group (p < 0.05). The levels of C and EC in the Bt1 treatment showed no significant changes, whereas both contents increased in the Bt2 group. Overall, the effects of different treatments on the quality components of tea leaves varied based on the spraying intensity, with the Bt1 treatment demonstrating a more pronounced effect, indicating that moderate Bt spraying is beneficial for improving tea quality.

In terms of enzyme activity (Figure 1B), the content of polyphenol oxidase (PPO), peroxidase (POD), and malondialdehyde (MDA) in tea leaves significantly increased after Bt treatment compared to the CK group (p < 0.05), with Bt2 showing higher levels than Bt1. Conversely, the content of superoxide dismutase (SOD) significantly decreased (p < 0.05). In summary, the antioxidant enzyme activity in tea leaves after Bt spraying showed a strong dependency on the varying intensities of Bt application. This indicates that increased spraying intensity may induce stress in tea leaves.

3.2. Effect of Bt Spraying on Phyllosphere Microbial Diversity in Tea Plants

The alpha diversity index provides insights into the richness and diversity of microbial communities. Boxplots illustrating the richness index (Chao1) and diversity index (Simpson) are shown in Figure 2A,B. Comparatively, the Bt1 treatment did not show significant differences in either the richness (Chao1 index) or diversity (Simpson index) of the endophytic and epiphytic bacterial communities on the tea leaves compared with the CK group. However, treatments that varied in intensity significantly reduced the richness index (Chao1, p < 0.05) of the fungal community on the leaves. Principal coordinate analysis (PCoA) performed at the operational taxonomic unit (OTU) level further demonstrated that there was a clear distinction between the microbial communities of the CK group and those from the Bt treatment, indicating a significant impact of Bt spraying on the structural composition of the bacterial and fungal communities in tea leaves (Figure 2C,D). In conclusion, while the application of Bt had minimal impact on the overall diversity of the phyllosphere microbiome, it significantly influenced the community structure of both bacteria and fungi present on the tea leaves.

Further analysis of the phyllosphere microbiome’s taxonomic composition at the phylum level, focusing on taxa with a relative abundance of greater than 1%, revealed distinct patterns within the bacterial community (Figure 2E,F). In the CK group, Actinobacteria was the predominant phylum, while other bacterial phyla appeared to be relatively sparse. Conversely, the application of the Bt1 and Bt2 treatments led to a significant increase in the relative abundance of Proteobacteria and Firmicutes. In terms of the fungal community, Ascomycota and Basidiomycota were the primary constituents. Ascomycota exhibited a pronounced dominance in the CK group; however, its relative abundance significantly declined in both Bt1 and Bt2 treatments. In contrast, the abundance of Basidiomycota significantly increased in both treatments. These results indicate that Bt application not only alters the overall composition of microbial communities but also affects the relative abundances of specific taxa within the phyllosphere.

3.3. Effect of Bt Spraying on the Symbiotic Microbial Network Within Tea Leaves

To explore whether the observed changes in the phyllosphere microbiome composition were correlated with alterations in microbial interactions, we conducted a co-occurrence network analysis of the epiphytic and endophytic microbial communities in tea plant leaves (Table S2). The results for the bacterial community (Figure 3A) indicated that the control (CK) group exhibited 112 nodes, 766 edges, and an average degree of 6.83. In the Bt1 treatment, the number of nodes slightly decreased to 105, while the number of edges increased to 1077, resulting in a higher average degree of 10.25 compared to the CK group. In the Bt2 treatment, the number of nodes rose to 113, while the number of edges was 957, leading to an average degree of 8.47 (Figure 3A). These findings indicate a notable enhancement in the connectivity of the bacterial community, particularly under the Bt1 treatment, where the bacterial network’s connectivity was at its highest compared with the CK group, characterized by an increased number of edges and a greater average degree, indicative of closer microbial interactions under this treatment.

For the fungal community, the CK group displayed 248 nodes, 1913 edges, and an average degree of 7.714. In the Bt1 treatment, the number of nodes slightly decreased to 229, while the number of edges increased to 1993, resulting in an average degree of 8.70. In the Bt2 treatment, the number of nodes further decreased to 246, with the number of edges remaining at 2027 and the average degree increasing to 8.23 (Figure 3B). Similar to the bacterial community, the fungal network demonstrated enhanced connectivity following Bt spraying, especially under the Bt2 treatment, where the average degree reached its highest value, indicating more complex interaction among fungi. In conclusion, our analysis revealed that the spraying of Bt significantly enhanced both the connectivity and the complexity of the phyllosphere microbiome in tea plants.

3.4. Effect of Bt Spraying on Key Phyllosphere Microorganisms in Tea Leaves

Further, key phyllosphere microbiomes under Bt spraying were screened using a random forest model (Table S3). For the analysis of endophytic bacteria (Figure 4A,B), Bt treatment resulted in a significant increase in the abundance of key genera, including Burkholderia-Caballeronia-Paraburkholderia, Methylobacterium-Methylorubrum, Pseudomonas, Rhodopseudomonas, and Novosphingobium (p < 0.05). Conversely, the relative abundances of Ellin6067 and Promicromonospora were notably decreased following the treatment. Similarly, among the epiphytic bacteria (Figure 4C,D), the relative abundance of genera such as Paracoccus, Pseudomonas, Methylobacterium, Bacillus, and Sphingomonas demonstrated a significant increase post-Bt treatment (p < 0.05). In contrast, the genera Aquabacterium and Bosea showed a significant decrease in abundance (p < 0.05). These findings indicate that Methylobacterium, Sphingomonas, Pseudomonas, Rhodopseudomonas, and Novosphingobium comprise key phyllosphere microbial groups that are enriched in response to Bt spraying.

In terms of the endophytic fungi (Figure 5A,B), the application of Bt led to a significant reduction in the abundance of potential pathogenic fungi such as Aspergillus, Pseudocercospora, and Phyllosticta (p < 0.05). Conversely, beneficial fungi such as Humicola were notably enriched in the Bt-treated group. For the epiphytic fungi (Figure 5C,D), the abundance of Humicola showed an upward trend following Bt treatment, whereas the genus Sphaeropsis was significantly inhibited.

3.5. Effect of Bt Spraying on the Functional Characteristics of Key Bacterial and Fungal Communities in the Tea Plant Phyllosphere

Further annotation of the phyllosphere bacterial metabolic functions was performed using the PICRUSt-KEGG (Kyoto Encyclopedia of Genes and Genomes) database. The results revealed that bacterial metabolic functions are primarily categorized into amino acid metabolism, carbohydrate metabolism, secondary metabolism, and photosynthesis metabolism (Figure 6A, Table S4). As shown in Figure 6, the biosynthesis of phenylalanine, tyrosine, and tryptophan, along with the metabolism of alanine, aspartate, and glutamate, exhibited a significant increase in the Bt treatment groups (p < 0.05). Furthermore, the biosynthesis of lysine and the branched-chain amino acids—valine, leucine, and isoleucine—also showed significant enhancement in response to Bt treatment. Additionally, the production of flavonoids under the category of secondary metabolism was significantly enhanced in the Bt-treated groups (p < 0.05), indicating that Bt treatment effectively promoted the production of secondary metabolites.

In terms of carbohydrate metabolism, the ructose and mannose metabolism, as well as galactose metabolism, were higher in the Bt treatment group compared to the CK group, suggesting that Bt treatment may enhance the efficiency of carbon source utilization. In the metabolic process related to photosynthesis, the phosphotransferase system (PTS) was found to be elevated after Bt treatment, potentially indicating its role in microbial regulation of plant metabolism. Overall, Bt treatment significantly enhanced the amino acid metabolism and secondary metabolism functions of the microorganisms associated with tea leaves, while also promoting carbohydrate metabolism and pathways related to photosynthesis. This enhancement may be associated with the observed effects of Bt treatment in improving the stress resistance and growth of tea plants.

Functional prediction of the fungal community in tea leaf samples, assessed using the FUNGuild database (Figure 6B, Tables S3 and S4), revealed the categorization of fungal ecological functions into three types: pathotroph, saprotroph, and symbiotroph. The results showed that Bt treatment significantly reduced the relative abundance of pathotrophs (p < 0.01). In contrast, the impact on saprotrophic fungi was relatively minor; however, there was an observed overall increasing trend in their abundance (p < 0.05). Conversely, Bt treatment significantly increased the abundance of symbiotrophs (p < 0.01), particularly mycorrhizal fungi, including ectomycorrhizal and endomycorrhizal fungi, which are known to form beneficial symbiotic relationships with plants.

3.6. Correlation of Key Phyllosphere Microorganisms with Tea Quality and Enzyme Activity Following Bt Spraying

The relationship between key differential bacterial genera and tea leaf quality, as well as enzyme activity, was further elucidated through Spearman correlation analysis (Figure 7A) and redundancy analysis (RDA) (Figure 7B). The RDA results indicate that the first two axes, RDA1 and RDA2, collectively accounted for 96.3% of the variation in the bacterial community and 90.78% of the variation in the fungal community. Regarding the bacteria genera, Burkholderia-Caballeronia-Paraburkholderia, Bacillus, Pseudomonas, Rhodopseudomonas, and Novosphingobium exhibited significant negative correlations with caffeine content and peroxidase (POD) activity (p < 0.05). Conversely, these genera showed significant positive correlations with free amino acids (AAs), flavonoids, soluble sugar, and other tea leaf quality indicators. Among the key differential fungi under treatment, some were significantly negatively correlated with POD, caffeine, and flavonoids (p < 0.05), while most other differential fungi showed positive correlations with AAs and soluble sugar. In summary, after Bt spraying, the changes in antioxidant enzyme activity and non-volatile metabolites of the tea plant were closely related to the community structure and function of both the bacteria and the fungi.

4. Discussion

Bt biopesticides, as widely used environmentally friendly pesticides, have a significant effect on resisting pests and diseases [9]. The phyllosphere microbiome, which is an important component of the plant microbiome, plays a crucial role in the health of tea plants and the quality of tea leaves. However, the regulatory effects of Bt pesticides on the phyllosphere microbiota and the response mechanisms of phyllosphere microbial communities are not well understood. Previous studies have shown that the substances within tea leaves are influenced by various factors, including environmental factors and agricultural practices [31,32]. The direct application of Bt pesticides to the surface of tea leaves is likely to affect the quality of the tea.

This study found that Bt treatment significantly increased the content of free amino acids, flavonoids, and soluble sugars, as well as catechins like EGCG and EGC in the leaves (Figure 1). Previous studies have shown that flavonoids can enhance the resistance of tea plants by inhibiting pathogen growth and scavenging reactive oxygen species (ROS) [33,34]. EGCG, a dominant antioxidant during the development of tea tree buds, plays a potentially key role in maintaining the stability of the leaf-associated microbiome [35,36]. It is worth noting that, with an increase in spraying frequency, the levels of SOD and POD in tea leaves under the Bt2 treatment decreased compared to those in Bt1, while the MDA content significantly increased. SOD, POD, and MDA are closely related to the stress resistance of tea plants [37]. The observed changes suggest that, under the conventional spraying frequency (Bt1), Bt as a biopesticide may induce mild stress in tea plants, leading to elevated SOD and POD activities. These enzymes help maintain redox balance by scavenging ROS, ultimately enhancing the plant’s stress resistance [38]. However, excessive Bt application (Bt2) may exceed the tolerance threshold of tea plants, causing excessive accumulation of ROS, inhibition of antioxidant enzyme activities, and a significant increase in MDA content. Therefore, the possibility of ecological risks arises from excessive Bt application [39].

The microbial communities in different parts of the tea plant are closely related to various metabolic processes. The leaf-associated microbiome, which is directly influenced by Bt spraying, plays a significant regulatory role in the interaction between Bt and the tea plant by modulating metabolic activities and enhancing phytochemical profiles. This interaction underscores the importance of maintaining a healthy microbiome, as it can significantly influence the overall health and productivity of the tea plants.

High-throughput sequencing was used to analyze the leaf-associated microbial community structure of tea plants under different Bt spray intensities. The results showed that the bacterial and fungal community structures of the Bt1 and Bt2 treatments were significantly different from those of the control treatment. However, there were no significant differences in bacterial alpha diversity indices among the three treatments, while the fungal diversity in the Bt treatments was lower than in the control. A previous study suggested that the function of microbial ecosystems primarily depended on functional diversity rather than taxonomic diversity [40], which implies that the changes in leaf contents under Bt spraying may not be directly related to the changes in bacterial/fungal community taxonomic diversity but are more likely mediated and regulated by the leaf-associated functional microbial communities. Notably, both the modularity and the complexity of the microbial co-occurrence network increased under Bt treatment. Modularity refers to the ecological niches within a microbial community [41]. The enhanced interactions among key phyllosphere microorganisms under Bt application may indicate a strengthened symbiotic relationship. This suggests that the microbial ecosystem is becoming more stabilized and cooperative, which may contribute to improved tea plant health.

Further bacterial community analysis revealed that Bt spraying significantly increased the abundance of Proteobacteria and Firmicutes in the leaf-associated microbiome (Figure 2E). Among these, the abundance of key functional genera, such as Burkholderia-Caballeronia-Paraburkholderia, Pseudomonas, Sphingomonas, and Methylobacterium, showed a synchronous increase (Figure 4A,B). Previous studies have indicated that these microbial communities participate in the nitrogen metabolism regulation of tea plants through multiple pathways. For instance, Burkholderia efficiently utilizes amino acids as nitrogen sources to drive organic nitrogen cycling [42]; Pseudomonas enhances nitrogen fixation and ammonia assimilation [43], which boosts the ammonia nitrogen levels in tea plants. Other studies have shown that Methylobacterium and Sphingomonas have a mutualistic relationship and promote the conversion of ammonia nitrogen to free amino acids [44,45]. Functional annotation of the bacterial community also revealed that Bt treatment significantly enhanced the amino acid metabolism and secondary metabolism functions of leaf-associated microorganisms. This suggests that the microbial-mediated functional transformation process may regulate the synthesis of free amino acids through ammonia nitrogen effects [46]. Previous studies have demonstrated that nitrogen form conversion driven by functional microorganisms can promote the accumulation of free amino acids by activating the tea plant enzyme system, and that nitrogen levels impact the activity of key enzymes in tea leaf nitrogen metabolism and non-structural carbohydrates [47]. Correlation analysis also revealed a significant positive correlation between these functional microbial communities and the content of free amino acids. Therefore, Bt spraying can enrich functional microbial communities such as Methylobacterium and Sphingomonas, promoting the accumulation of free amino acids and significantly affecting the quality of tea plant leaves.

Bt has a significant effect on tea plant diseases, with many research findings in this area. In this study, regarding the fungal community, Bt treatment significantly reduced the abundance of plant pathogens such as Aspergillus and Phyllosticta (Figure 5), while enriching genera with biocontrol potential, such as Teichospora and Humicola [48,49,50,51]. Functional predictions further indicated that the proportion of plant pathogen-related functions significantly decreased under Bt treatment (Figure 6B). Previous studies have shown that the accumulation of key antioxidant components such as EGCG and EGC in tea leaves can form an allelopathic barrier against pathogen invasion by regulating the leaf-associated microbial interaction network [52]. Notably, Bt treatment increased the complexity and modularity of the microbial community network compared to the control group (Figure 3). Bt treatment enhanced the interactions between key microbial groups in the leaf environment, and higher modularity values indicate that the structure and symbioses of the microbial ecosystem were promoted. This indirectly increased the tea plant’s resistance to pathogens. Therefore, Bt’s ability to resist pathogens may not only be attributed to its direct inhibitory effect on pathogens but also to the enhanced stability of the leaf-associated microbial community, which indirectly aids in resisting pathogen invasion [53,54]. In summary, while Bt spray significantly inhibits tea plant pathogens, it also significantly affects tea quality by mediating the content of amino acids and catechins.

5. Conclusions

This study reveals the regulatory effects of Bacillus thuringiensis pesticide on the leaf-associated microbial community of tea plants and its potential mechanisms. Bacillus thuringiensis spraying significantly increased the content of important metabolites, such as free amino acids, flavonoids, soluble sugars, and catechins in the tea leaves. Meanwhile, excessive application of Bacillus thuringiensis poses certain ecological risks, as it can lead to a decrease in superoxide dismutase and peroxidase activities in tea leaves. Furthermore, Bacillus thuringiensis treatment significantly altered the structure of the leaf-associated microbial community, increased the abundance of key functional genera, and indirectly enhanced the tea plant’s disease resistance by improving the network complexity and modularity of the microbial community. These results offer new insights into the ecological role of Bacillus thuringiensis-based biopesticides and may help optimize their practical application strategies in agricultural systems. However, the interactions between plants and microorganisms are highly complex. Future studies should include field validations across different tea cultivars, combined with transcriptomic and other omics-based approaches, to further elucidate the underlying mechanisms.