Impact of Tebuconazole On the Development and Symbiotic Microbial Communities of Pardosa Pseudoannulata

Abstract

Tebuconazole is a widely used triazole fungicide to control fungal diseases. While there have been reported side effects on non-target arthropods, its ecological risks to natural enemies remain poorly understood. In this study, we evaluated the developmental toxicity and symbiotic microorganism responses of the wolf spider Pardosa pseudoannulata, an important predator in rice ecosystems, following exposure to tebuconazole. The results indicated that tebuconazole did not significantly increase the mortality rate of spiderlings; however, it did lead to a significant decrease in spiderling body weight, as well as the length and width of the carapace. High-throughput sequencing of the 16S rRNA gene V3–V4 regions and the ITS region revealed that tebuconazole significantly reduced bacterial diversity indices in the short term, with a gradual recovery over time. In contrast, the impact on the fungal community was continuous and irreversible, with a significant decrease in the Shannon index observed after 15 days. At the genus level, the relative abundances of Cupriavidus and Staphylococcus in the bacterial community decreased significantly after tebuconazole exposure, while Stenotrophomonas increased. In the fungal community, Fungi_gen_Incertae_sedis decreased significantly, and Simplicillium increased. Our findings highlight the ecological risks of fungicide exposure to beneficial predators and underscore the importance of considering symbiotic microbiota in pesticide risk assessments.

Supplementary Information

The online version contains supplementary material available at 10.1007/s00248-025-02600-6.

Introduction

Tebuconazole (TEB) is a commonly utilized triazole fungicide, which plays a significant role in controlling various fungal diseases across diverse agricultural sectors [1]. TEB acts by inhibiting the activity of lanosterol 14α-demethylase, interfering with the biosynthesis of ergosterol, and leads to the inhibition of cell growth or cell death in fungi [2, 3]. As a foliar spray, it effectively controls fungal pathogens in rice and wheat, including powdery mildew and rust species, thereby increasing yields [4].

Pardosa pseudoannulata (Araneae: Lycosidae) serves as a crucial predator in rice ecosystems and is widely distributed across Asia [5, 6]. This species effectively preys on both large nymphs and adults of N. lugens in rice fields [7]. Interestingly, in addition to the effectiveness of fungal diseases, tebuconazole also indirectly improves the control effect of pests. Studies have found that tebuconazole not only significantly increased the mortality of Nilaparvata lugens (Hemiptera: Delphacidae), but also had a negative impact on its body weight, digestive enzyme activity, and reproductive capacity [8]. Tebuconazole also causes negative impacts on a variety of animals [9, 10]. For example, the establishment, nesting period, and fecundity of bees exposed to a mixture of tebuconazole and acetamiprid were significantly reduced [11]. From a biological control perspective, the presence of P. pseudoannulata provides significant advantages in managing N. lugens populations [12]. However, the effects of tebuconazole on this natural enemy have not been reported yet.

Studies suggest that the insecticidal mechanism of tebuconazole against N. lugens was possibly achieved by disrupting microbial homeostasis [13]. Tebuconazole significantly influenced the diversity of symbiotic fungi of N. lugens, as well as the relative abundance of saprophytic and pathogenic microorganisms [14]. The number of symbionts and the expression level of Noda were significantly diminished in the N. lugens that received treatment with tebuconazole [8, 15]. Furthermore, the administration of tebuconazole in mice led to the promotion of cognitive impairment and colitis through the modulation of gut microbiota and the microbe-gut-brain axis [16]. Research has demonstrated that symbiotic organisms are vital for the host’s life processes, including growth, development, and reproduction [17, 18]. Over extended evolutionary periods, these microorganisms have formed a mutually advantageous relationship with their host [19]. Furthermore, research on the symbiotic microorganisms of P. pseudoannulata has primarily concentrated on the impacts of the environmental pollutant cadmium on its epidermis and intestinal bacteria [20, 21]. To date, studies investigating the effects of tebuconazole on the symbiotic bacteria and fungi of P. pseudoannulata have not been conducted.

Therefore, we conducted a preliminary investigation into the survival and development of P. pseudoannulata spiderlings in the presence of tebuconazole. Subsequently, we assessed the effects of tebuconazole exposure on the microbial community of symbiotic bacteria and fungi by 16S rRNA and ITS analysis. The results of this study provide critical data for evaluating the ecological risks associated with tebuconazole and hold significant practical value in coordinating the synergistic effects of chemical and biological control.

Materials and Methods

Spider Collection and Rearing

Pardosa pseudoannulata were collected in October 2024 from a fungicide-free rice field in Wuhan, Hubei Province, China. The spiders were reared in a climate-controlled room, where the conditions were maintained at 26 ± 1 °C, 65 ± 5% relative humidity (RH), and a 16 h/8 h light–dark cycle. Mature spiders were randomly selected for mating. The hatched spiderlings were reared individually in glass tubes (20 mm diameter, 100 mm height) lined with a moist sponge at the bottom of each tube for subsequent experiments.

Tebuconazole Treatment and Biological Characterization

Tebuconazole (125 mg/L) was applied at the concentration recommended by the China Pesticide Information Network (http://www.chinapesticide.org.cn/). The bioassay was performed utilizing the dipping technique as outlined in previous research [22]. In summary, carbon dioxide was used to anesthetize the second instar spiderlings at a flow rate of 5 L/min for a duration of 15 s. Subsequently, the spiderlings were immersed in the insecticide solution for 20 s. The control group was treated with pure water without tebuconazole. After treatment, the spiderlings were placed on filter paper to eliminate the excess solution, and once they regained consciousness, each spiderling was individually transferred into petri dishes and fed N. lugens. The spiderlings were provided with four N. lugens every 2 days, starting from the second day post-treatment.

Each treatment group consisted of 45 spiderlings. Survival and molting were recorded daily. On the 14th day, body weight was measured using a BT1251 scale (Sartorius Scientific Instruments, China), and carapace length and width were assessed with a Leica 205 C digital microscope.

Sample Collection, DNA Preparation, and PCR Amplification

After 1 and 15 days of tebuconazole treatment, the spiderlings were collected and surface sterilized with 75% ethanol for 20 s and then rinsed three times with sterile water. The control group was treated as above, with six replicates of each treatment. Each replicate was composed of 10 individuals.

Microbial community genomic DNA was obtained utilizing the CretMag™ Power Soil DNA Kit (CretBiotech, China) following the manufacturer’s instructions. The DNA was checked on 1.2% agarose gels, and quantity was measured on a NanoDrop 2000 UV–vis spectrophotometer (Thermo Scientific, Wilmington, CA, USA). The amplification of the bacterial 16S rRNA gene’s V3–V4 variable regions was conducted using the primers 338 F (5′-ACTCCTACGGGAGGCAGCAG-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′) [23]. To analyze fungal diversity, universal primers ITS1F (5′-CTTGGTCATTTAGAGGAAGTAA-3′) and ITS2R (5′-GCTGCGTTCTTCATCGATGC-3′) were employed to amplify the ITS of the fungal ribosomal operon [24].

A total of 50 µL of PCR reaction mixture containing 25 µL of 2 × Es Taq MasterMix, 2 µL of each primer, 2 µL of template DNA, and ddH₂O was added to the mixture. The following parameters were used in the PCR cycle: denaturation at 94 °C for 2 min, followed by 30 cycles of 30 s at 94 °C, 30 s at 55 °C, and 30 s at 72 °C. For the last cycle, the elongation time was extended to 10 min at 72 ° C. The PCR product was extracted from 2% agarose gel (Fig. S1-S2) and purified using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, USA) according to the manufacturer’s instructions and quantified using Quantus™ Fluorometer (Promega, USA).

High-Throughput Sequencing and Bioinformatic Processing

VAHTS® Universal Plus DNA Library Prep Kit for MGI V2 (Novizan, China) was used to build the library: (1) splinter link, (2) the use of magnetic bead screening to remove the joint self-contiguous segments, (3) enrichment of library templates by PCR amplification, (4) the use of magnetic beads to recover PCR products, and (5) cyclization of PCR products. Sequencing is performed using DNBSEQ-G99 PE300 platform of MGI (BioWefind Biotechnology Co., LTD).

The raw sequencing reads were demultiplexed, quality-filtered by fastp version 0.20.0 [25] and merged by FLASH version 1.2.7 [26] with the following criteria: (i) the 300-bp reads were truncated at any site receiving an average quality score of < 20 over a 50-bp sliding window, and the truncated reads shorter than 50 bp were discarded; reads containing ambiguous characters were also discarded; (ii) only overlapping sequences longer than 10 bp were assembled according to their overlapped sequence. The maximum mismatch ratio of the overlap region is 0.2. Reads that could not be assembled were discarded; (iii) samples were distinguished according to the barcode and primers, and the sequence direction was adjusted, with exact barcode matching and two nucleotide mismatches in primer matching.

Operational taxonomic units (OTUs) with 97% similarity cutoff [27, 28] were clustered using UPARSE version 7.1, and chimeric sequences were identified and removed. The 16S rRNA and ITS were classified for each OTU representative sequence using Silva v138 and the Ununite database, respectively, using a confidence threshold of 0.7 [29].

Statistical Analysis

The statistical analyses were conducted using R software, version 4.4.3 [30]. To assess the normality of the data and the homogeneity of variances, both the Shapiro–Wilk and Levene’s tests were utilized, complemented by visual inspections through Q–Q plots. The comparison of survival curves was performed using the log-rank test (Mantel-Cox). T-test was employed to analyze body weight, carapace length, and width. Based on the OTUs information, rarefaction curves and alpha diversity indices including observed Shannon index, Shannon index, Chao1 richness, and ACE index were calculated with Mothur v1.30.1 [31]. The similarity among the microbial communities in different samples was determined by principal coordinate analysis (PCoA) based on Bray–Curtis dissimilarity using Vegan v2.5–3 package.

Results

Tebuconazole Effect on the Development of Spiderlings

The effect of the tebuconazole on the development of spiderlings is shown in Fig. 1. The mortality in the tebuconazole treatment was higher than that in the control group; however, this difference was not statistically significant (Mantel-Cox test, χ² = 3.147, p = 0.076) (Fig. 1A). In comparison to the control group (2.25 ± 0.79 mg), the weight of the spiderlings at 14 days after tebuconazole treatment (1.76 ± 0.59 mg) was significantly reduced (p = 0.039) (Fig. 1B). The carapace length in the tebuconazole group (1.319 ± 0.161 mm) was statistically significantly different from that in the control group (1.328 ± 0.084 mm) (Fig. 1C). Similarly, the carapace width was significantly lower in the tebuconazole group (1.005 ± 0.111 mm) compared to the control group (1.059 ± 0.070 mm) (Fig. 1D). These results indicate that the application of the fungicide tebuconazole negatively affects the development of spiderlings of P. pseudoannulata.

Fig. 1.

Biological characteristics of Pardosa pseudoannulata spiderlings after tebuconazole exposure: A survival curves of spiderlings, B body weight, C carapace length, and D carapace width. The level of significance for the tebuconazole treatment compared to the control was set at *p < 0.05 and ***p < 0.001; CK, control; TEB, tebuconazole

The Diversity Analysis of the Bacterial and Fungal Community

To investigate the effect of tebuconazole on the symbiotic microbial community and its structure in P. pseudoannulata spiderlings, we sequenced the 16S rRNA gene V3-V4 variable regions and ITS variable regions at 1 and 15 days after treatment, respectively. After preliminary quality control, a total of 5,090 bacterial operational taxonomic units (OTUs) (Fig. 2A) (Table S1) and 1,104 fungal OTUs (Fig. 2B) were identified from 48 samples based on 97% sequence similarity (Table S2).

Fig. 2.

Venn diagram demonstrating the quantities of common and unique microorganism species at the OTU level between A bacteria and B fungi. Each measurement contains six replicates; CK, control; TEB, tebuconazole

For the bacterial community, the Shannon index (p = 0.025), Simpson index (p = 0.037), and Chao1 index (p = 0.037) were significantly lower on the first day of tebuconazole treatment compared to the control group (Table 1). However, after 15 days of treatment, no significant differences in bacterial diversity indices were observed (Table 1). This indicates that tebuconazole significantly impacts the symbiotic bacterial community of P. pseudoannulata spiderlings in the short term; however, this impact appears to gradually recover over time.

Table 1.

Characteristics of microbial community alpha diversity in spiderlings

Sample	Shannon	Simpson	Chao1	ACE
Bacterial community
CK_1d	4.79 ± 1.42	0.92 ± 0.06	1051.15 ± 167.92	1098.01 ± 151.74
TEB_1d	3.31 ± 0.44	0.84 ± 0.05	912.32 ± 82.32	960.53 ± 88.05
CK_15d	2.95 ± 0.58	0.79 ± 0.07	750.32 ± 58.89	777.62 ± 49.22
TEB_15d	2.93 ± 0.50	0.84 ± 0.09	706.27 ± 87.52	700.15 ± 94.84
Fungal community
CK_1d	3.24 ± 0.71	0.88 ± 0.07	264.62 ± 72.62	262.34 ± 68.33
TEB_1d	2.74 ± 0.41	0.84 ± 0.07	189.99 ± 37.16	193.17 ± 36.60
CK_15d	2.16 ± 0.50	0.79 ± 0.09	149.90 ± 23.37	152.28 ± 27.05
TEB_15d	1.55 ± 0.43	0.67 ± 0.12	124.35 ± 32.23	127.78 ± 31.93

CK control, TEB tebuconazole

For the fungal community, compared with the control group, all indices decreased on the first day after treatment but there was no significance. Only on the 15th day, the Shannon index of juvenile spiders after tebuconazole treatment decreased significantly (p = 0.037) (Table 1). The results indicate that tebuconazole has a continuous and irreversible impact on the symbiotic fungal community of spiderlings.

The Community Structure of the Bacterial and Fungal Community

The dissimilarity of bacterial and fungal community structures in spiderlings was compared using PCoA plots based on the Bray–Curtis distance. For the bacterial community, the PCoA1 and PCoA2 represented 46.79% and 15.24% of the data variation on the first day after treatment, respectively (R² = 0.169, p = 0.03) (Fig. 3A). Significant changes were observed in the bacterial composition between the tebuconazole treatment group and the control group. On the 15th day, the PCoA1 and PCoA2 values were observed to be 37.2% and 27.38%, respectively, and there was no significant difference in the microbial composition between the treatment group and the control group (R² = 0.151, p = 0.078) (Fig. 3B). These graphs indicate that following tebuconazole treatment, there is a significant alteration in the symbiotic bacterial community of spiderlings. However, this change appears to gradually recover over time.

Fig. 3.

Principal coordinate analysis PCoA of symbiotic bacterial and fungal communities of Pardosa pseudoannulata spiderlings exposed to tebuconazole: A the PCoA of symbiotic bacteria at 1 day, B the PCoA of symbiotic bacteria at 15 days, C the PCoA of symbiotic fungi at 1 day, and D the PCoA of symbiotic fungi at 15 days. Each treatment group contains six replicates; CK, control; TEB, tebuconazole

For the fungal community, PCoA1 and PCoA2 on the first day accounted for 48.31% and 14.7% of the data, respectively (Fig. 3C). Compared with the control group, there was no significant difference in the fungal community treated with tebuconazole (R² = 0.106, p = 0.214). However, at the 15th day, the fungal community in the TEB treatment group changed significantly (R² = 0.628, p = 0.003) (Fig. 3D). This analysis indicates that the change of tebuconazole in the fungal community is continuous and irreversible.

Relative Abundances of Several Genera Were Changed by the Exposure of Tebuconazole

The relative abundances of the top 10 genera were compared across all the samples (Fig. 4). As for the bacterial constitution, on the first day, at the genus level, the samples primarily contained Cupriavidus, Staphylococcus, and Stenotrophomonas (Fig. 4A). Among them, the relative abundances of Cupriavidus and Staphylococcus decreased by 11.39% and 17.72%, respectively, after contacting TEB, while the abundance of Stenotrophomonas increased by 19.51% compared to the control group (Fig. 4A). On the 15th day following TEB treatment, the symbiotic bacterial community in spiderlings was predominantly composed of the genera Cupriavidus, Sphingomonas, and Enhydrobacter. The abundance of these genera in the TEB-treated group exhibited minimal variation compared to the control group (Fig. 4B).

Fig. 4.

Tebuconazole affects the relative abundance of symbiotic bacteria and fungi in spiderlings at the genus level: A the relative abundance of symbiotic bacteria at 1 day, B the relative abundance of symbiotic bacteria at 15 days, C the relative abundance of symbiotic fungi at 1 day, and D the relative abundance of symbiotic fungi at 15 days. Each color block corresponds to a genus, and each treatment group contains six replicates: CK, control; TEB, tebuconazole

At the fungal level, on the first day following tebuconazole treatment, the predominant genera identified were Fungi_gen_Incertae_sedis, Fusarium, and Aspergillus, with minimal variation among them (Fig. 4C). However, by the fifteenth day post-treatment, a significant difference in the dominant genera was observed. The top three genera included Fungi_gen_Incertae_sedis, Simplicillium, and Penicillium. In comparison to the control group, the abundance of Fungi_gen_Incertae_sedis decreased by 71.17%, whereas the relative abundance of Simplicillium increased by 67.17% (Fig. 4C).

Discussion

Fungicides initially developed for controlling phytopathogenic microorganisms [32, 33] have recently been recognized for their unexpected insecticidal properties. Tebuconazole (TEB), a systemic triazole fungicide, has garnered attention in agricultural practices for its potential effects not only on plant pathogens but also in assisting insecticides with pest control [14, 34]. While primarily targeting fungal pathogens through inhibition of ergosterol biosynthesis [2], emerging evidence highlights its role as a pest control adjuvant. Research has demonstrated that tebuconazole exhibits significant insecticidal activity against the brown planthopper (BPH) [8, 10, 13]. Following ingestion of the fungicide, there is a marked increase in the mortality rate of the brown planthopper, alongside detrimental effects on its body weight, digestive enzyme activity, and reproductive capacity [8]. However, the ecological impact of these insecticide effects extends beyond the target pests to include their natural enemies. In this study, we assessed the toxic effects of tebuconazole on the natural enemy of the brown planthopper, P. pseudoannulata. The present study revealed that tebuconazole did not significantly increase the mortality rate of spiderlings but resulted in a 22% decrease in body weight along with adverse effects on both carapace length and width. As a major predator of brown planthoppers in rice ecosystems [5], developmental impairments in P. pseudoannulata may compromise its biocontrol efficacy. Further ecological impacts necessitate verification through field studies.

The investigation into the microorganisms associated with P. pseudoannulata following exposure to tebuconazole revealed significant alterations in microbial communities, with distinct impacts observed on both bacterial and fungal populations. Day one post-exposure, the Shannon index of the bacterial community exhibited a 32% reduction; however, it returned to control group levels by day 15. This suggests that the bacterial community may have some resilience to tebuconazole exposure, although further studies are needed to determine the long-term stability of these communities. A parallel pattern was observed in carbendazim-treated soils; bacterial communities recovered within 7 days, whereas fungal diversity remained depressed [35]. Similarly, the soil fungal Shannon diversity decreased steadily after 30 days of tebuconazole exposure, while bacterial diversity transiently fluctuated [1]. These independent observations support the hypothesis that triazole fungicides exert stronger and longer-lasting disturbances on fungal symbionts than on bacterial associates.

Genus-level analyses showed that short-term exposure to TEB significantly altered the structure of the spider bacterial community. For example, Cupriavidus and Staphylococcus were reduced by 11.4% and 17.7%, respectively, which may have led to limited energy acquisition by the host, consistent with the observed developmental inhibition. The 19.5% increase in the abundance of the genus Stenotrophomonas may be related to the subsequent restoration of the bacterial community. Previous research has indicated that the metabolic activities of Stenotrophomonas can facilitate the decomposition of pesticides [36]. For example, Stenotrophomonas pavanii DJL-M3, immobilized on rice-husk biochar, became dominant under carbendazim stress and significantly reduced fungicide residues in the rice rhizosphere [37]. These reports suggest that Stenotrophomonas may act as a keystone taxon facilitating bacterial community resilience following triazole exposure.

In contrast, the fungal community exhibits an irreversible decline in abundance, as evidenced by a 41% reduction in the Shannon index at 15 days. This disparity may be attributed to the targeted mechanism of TEB, which inhibits the ergosterol synthase specific to fungi [3], preferentially destroying the stability of the fungal community. The observed shifts in fungal genera, such as the significant increase in Simplicillium and decrease in Fungi_gen_Incertae_sedis, may have profound implications for the spider’s ability to maintain its ecological functions. Research has demonstrated that the genus Simplicillium is pathogenic to insects. A newly isolated strain, Simplicillium lanosoniveum, exhibits significant entomopathogenic potential, effectively suppressing all developmental stages of the rugose spiraling whitefly (RSW) [38]. The fungal entomopathogen community surrounding Gibellula infected spiders was analyzed using ITS amplicon sequencing. The results revealed that members of the genus Simplicillium from the family Cordycipitaceae, Metarhizium from Clavicipitaceae, and Purpureocillium and Polycephalomyces from Ophiocordyceps predominated [39].

Previous studies on the symbiotic microorganisms of P. pseudoannulata have primarily focused on the impact of environmental stressors, such as cadmium pollution, on the diversity of its gut microbiota [20, 40, 41]. This study reveals the dynamic response patterns of the spider’s symbiotic bacterial and fungal communities mediated by tebuconazole (TEB) through exposure experiments. While current research primarily focuses on the effects of tebuconazole on P. pseudoannulata microorganisms, the inhibition of gene expression may represent another crucial mechanism. Future studies will integrate host gene expression profiling with microbial functional genomics to elucidate the transboundary toxicity regulatory network of tebuconazole.

Conclusion

In the current study, we observed that the triazole fungicide tebuconazole did not significantly affect the survival of P. pseudoannulata spiderlings; however, it significantly inhibited their growth and disrupted their symbiotic microbial community. Bacterial diversity exhibited transient declines but recovered by day 15, while fungal communities underwent irreversible structural shifts, including a notable 67.17% increase in the abundance of Simplicillium, indicating persistent dysbiosis. These disruptions, particularly in fungal symbionts that are critical for host fitness, are likely to contribute to developmental inhibition in spiders. Given that P. pseudoannulata plays a vital role in pest control, the microbial imbalance induced by tebuconazole may indirectly compromise the efficacy of biological control in rice ecosystems. These findings underscore the importance of incorporating considerations of microbial symbiosis into agrochemical risk assessments and advocate for balanced pest management strategies to protect natural enemies and maintain ecosystem stability.

Supplementary Information

Below is the link to the electronic supplementary material.

Author Contribution

Peijie Cheng: Writing – original draft, Methodology, Investigation, Formal analysis, Writing – review & editing. Fengjie Liu: Writing – original draft, Methodology, Investigation, Formal analysis, Writing – review & editing, Funding acquisition. Lei Li: Visualization, Methodology, Formal analysis. Shuhan Wu: Validation, Investigation, Data curation. Wenjing Xiao: Investigation, Data curation. Qiong Zong: Writing – review & editing. Tao Liu: Writing – review & editing, Validation. Yu Peng: Conceptualization, Writing – original draft, Writing – review & editing, Supervision, Funding acquisition.

Funding

This study is supported by the Open Fund Project of Hubei Key Laboratory of Regional Development and Environmental Response [2024(C)002].

Data Availability

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found at https://www.ncbi.nlm.nih.gov/, PRJNA1237159.

Declarations

Competing Interests

The authors declare no competing interests.