Impacts of sulfur application on microbial communities and functional attributes in rubber plantation soil

Abstract

Elemental sulfur is widely used in fungicide applications to control crop diseases in agricultural systems, but its effects on soil microbial communities are largely unknown. In China, significant amounts of elemental sulfur are sprayed annually on rubber plantations to support crop performance. To investigate the effects of sulfur spraying on microbial diversity, composition, interactions, and functionalities in rubber plantation soil, soil samples from rubber plantations in Yunnan, South China, were collected, and bacterial and fungal communities were analyzed through high-throughput sequencing. Results showed that sulfur application did not alter the alpha diversity but the beta diversity of the soil bacterial community. Notably, sulfur disturbed the relative abundances of Chloroflexi and Planctomycetes. Certain bacteria (e.g., Bacillus and Sinomonas) thrived under sulfur treatment, influencing nutrient cycling. The ecological network analysis revealed enhanced bacterial and fungal interconnections. Sulfur application had a limited impact on microbial phenotypes and community functions, yet it inhibited sulfur compound respiration. These findings indicate that sulfur spraying can shift microbial community composition and influence nutrient cycling by favoring specific microbial groups. Despite its limited impact on microbial phenotypes, sulfur affects key metabolic processes, such as sulfur respiration, which are vital for soil health and microbial activity. The results highlight the need for sustainable sulfur management to optimize nutrient cycling and soil health on rubber plantations.

Introduction

Elemental sulfur is recognized for its efficacy against insect pests and diseases and is commonly used in fungicide applications to address issues like powdery mildew [1]. However, intensive use of elemental sulfur in agricultural soil can have profound impacts on soil microbial activity and the accessibility of soil micronutrients [2, 3]. Excessive accumulation of elemental sulfur in soil results in reducing cation exchange capacity, shifts in microbial community composition, and diminished nutrient availability [4, 5]. Additionally, the effects of sulfur extend to pH regulation and sulfate accumulation due to elemental sulfur oxidation [6, 7], closely linked to soil acidity levels [8, 9]. Understanding how sulfur influences the diversity, composition, and functional traits of soil microbial communities is crucial, especially when excessive sulfur amendments into agricultural soils occur due to human activities.

Hevea brasiliensis, commonly known as the rubber tree, is a vital source of natural rubber, supplying the essential material for latex coagulation and various rubber products. In regions with subtropical climates, such as China, the presence of powdery mildew poses a significant threat, potentially leading to rubber yield reductions of up to 45% [10]. To face this issue, sulfur spraying has emerged as the most effective method for controlling powdery mildew in rubber trees. Elemental sulfur, known for its cost-effectiveness and high efficacy, is the preferred pesticide among farmers. During the leaf-expansion phase of rubber trees in February and March, Elemental sulfur is sprayed two to three times annually to mitigate the problem. Historical data from rubber plantations in Xishuangbanna, Yunnan province, suggest an annual usage of 6,300 to 12,700 tons of sulfur powder for spraying, with an average application rate of 15 kg per hectare. Over time, as sulfur application accumulates throughout the rubber plantation’s developmental stages, noticeable changes occur in Yunnan’s soil total sulfur patterns and related soil microbial properties [11]. The total soil sulfur content negatively correlates with soil microbial respiration, indicating that elemental sulfur adversely affects soil microbial activity in rubber plantations [12]. However, the effects of sulfur spraying on the diversity, composition, and functional characteristics of microbial communities in rubber plantation soil have not been thoroughly investigated.

Thus, soil samples from rubber plantations situated in the Yunnan region were selected to investigate the effects of sulfur on the following aspects: (1) soil microbial diversity, structure, and composition of bacterial and fungal communities; (2) intraspecific and interspecific interactions within bacterial and fungal communities; and (3) microbial functionalities of these communities. Considering the extensive use of elemental sulfur in disease control for crops, the findings from this study would contribute to a better comprehension of the ecological risks stemming from its use within agroecosystems.

Materials and methods

Chemicals and spraying equipment

The sulfur powder, procured from Biaosheng Chemical Co., Ltd., Shanxi, China, is primarily composed of elemental sulfur (91%) and the additive talc powder (9%). Talc powder is commonly used as a dispersant in industrial applications and is generally considered inert with regard to microbial interactions. The spraying equipment utilized was the Guangyi 6HWF-20 backpack gasoline-powered sprayer, chosen to ensure uniform distribution of the powder within rubber plantations.

Experimental design and soil sampling

The experimental plantation (N103°98′, E22°58′), affiliated with Mahuangbao farm, is situated in Hekou County, Yunnan Province, China. This region experiences a tropical monsoon rainforest warm climate. The plantation is dedicated to rubber cultivation in a monoculture system. The planting duration is 15 years, and the soil is typical red soil. Prior to the study, informed consent was obtained from the farm owners. The site comprised two replicate plots (100 × 40 m) within the plantation area, designated as the sulfur treatment and the control. The plots were selected based on their similar soil properties, prior treatments, and environmental conditions to minimize variability between the two. Both plots were similarly affected by a powdery mildew outbreak in 2021, and sulfur spraying was carried out in March 2021 as per the guidelines for forecasting rubber tree powdery mildew [13]. The initial application of sulfur took place on March 10th, with a dosage of 15 kg per hectare, following standard practices commonly used in rubber plantations for managing powdery mildew. Subsequently, treatments with the same dosage were carried out at 7-day intervals, totaling three applications. Soil samples were collected on the 7th, 14th, 30th, and 60th days after the third application. At each sampling event, five 10 m × 10 m quadrats were selected from both control and treatment plots based on a randomized partitioning approach within the bare soil between rubber trees. In each quadrat, five soil samples (1 kg each) were collected from a depth of 30 cm using a soil auger. Before collection, any litter-fall or grass layer was carefully cleared. Plant debris, including roots and stones, were meticulously removed. Post-collection, each soil sample underwent sieving using a 2-mm sieve. The sieved soil was then quartered using the quartering method, and 10 g was subsampled, placed in sterile bags, and stored at -80 °C for subsequent DNA extraction.

DNA extraction and PCR amplification

Total microbial DNA from each 0.25 g soil sample was extracted using the HiPure Soil DNA Kit (Magen, Guangzhou, China), following the manufacturer’s instructions. The purified DNA was stored at -20 ℃ until a quality inspection could be conducted. The V3-V4 region of the bacterial 16 S rRNA gene was PCR-amplified using the forward primer 341 F (5’-CCTACGGGNGGCWGCAG-3’) and the reverse primer 806R (5’-GGACTACHVGGGTWTCTAAT-3’) [14]. Similarly, the ITS2 region of the fungal ITS RNA gene was amplified through PCR using the primer pairs ITS3_KYO2 (5’-GATGAAGAACGYAGYRAA-3’) and ITS4 (5’-TCCTCCGCTTATTGATATGC-3’) [15].

Sequence processing and bioinformatic analysis

The library sequencing was conducted using the Illumina Novaseq 6000 platform by Gene Denovo Biotechnology Co., Ltd (Guangzhou, China). The high-throughput sequences from specific 16 S/ITS regions were denoised utilizing DATA2 to generate amplicon sequence variants (ASVs) for each sample [16]. Bacterial and fungal taxonomic classifications were determined using the Silva 138 database and the Unite 8.0 database, respectively, with the Bayes classifier method [17]. The original raw sequence reads have been archived in the Sequence Read Archive Database at the National Center for Biotechnology Information (Accession Numbers: PRJCA019081).

Bioinformatic analyses and diagrams were executed using the Omicsmart online platform (http://www.omicsmart.com) to ensure the robustness and consistency of findings. Calculations encompassed alpha-diversity indices, namely Sobs, ACE, Shannon, and Shannon evenness indices. The statistical significance of discrepancies between the sulfur treatment and the control was evaluated via Welch’s t-test (P < 0.05). Principal coordinates analysis (PCoA) based on Bray-Curtis dissimilarity was adopted to visually depict shifts in microbial community composition. The evaluation of similarity in microbial community structures involved a multivariate PERMANOVA, using the Adonis function. Shifts with a P value less than 0.05 were considered statistically significant, and R² was used to assess the magnitude of these shifts. Detection of disparities in the abundance of bacterial genera between sulfur-treated and control groups was facilitated by integrating the linear discriminant analysis effect size (LEfSe) method with the Kruskal-Wallis Sum Rank test. Inference of co-occurrence patterns within bacterial and fungal communities was achieved using network analysis grounded in random matrix theory [18]. The annotation of bacterial and fungal functionalities relied on BugBase, PICRUSt2, Tax4Fun, and FAPROTAX databases accessible via the Omicsmart online platform. For comparative analysis, a one-way analysis of variance (ANOVA) was employed, with statistical significance set at P < 0.05.

Results and discussion

Sulfur spraying effects on microbial diversity and community dynamics in rubber plantation soils

The effects of sulfur spraying on bacterial and fungal communities in rubber plantation soils were analyzed. A total of 5,101,286 high-quality sequences were obtained from 40 soil samples after merging and filtering raw data of reads (Table S1). The rarefaction curves for bacteria and fungi (Fig. S1) indicated a sequencing coverage exceeding 0.99, ensuring the adequacy of depth for 38 samples. Evaluating alpha diversity provided insights into the variations in community composition among the samples (Fig. 1). To quantify species richness, diversity, and homogeneity of microbial communities, the Sobs, ACE, Shannon, and Simpson indices were employed [19]. The average values of alpha diversity indices for both bacteria and fungi are presented in Fig. 1. Over time, both the CK and sulfur-treated groups exhibited minor fluctuations in the four index values, implying a stable species richness state in the prokaryotic and eukaryotic microbial communities within the rubber plantations. Notably, the prokaryotic microbial communities displayed higher species richness compared to the eukaryotic counterparts, signifying the prevalence of prokaryotes in the rubber plantations. Such dominance has been frequently observed in nutrient-rich, cultivated soils [20, 21]. Furthermore, the alpha diversity indices did not differ significantly (P > 0.05) between the CK and sulfur-treated groups, indicating that sulfur spraying had no detectable effect on the overall microbial richness and evenness in these rubber plantations.

Fig. 1

Alpha diversity indices (Sobs, Ace, Shannon, and Simpson) for the soil microbial community following exposure to sulfur for 7, 14, 30, and 60 days. The letters “P” and “E” denote the prokaryotic and eukaryotic microbial communities, respectively. The letter “S” represents the application of sulfur spray. No statistical significance was found for discrepancies between the sulfur treatment and CK using Welch’s t-test (P < 0.05)

The PCoA and Adonis analysis were employed to assess the beta diversity, which captures the dissimilarity between samples (Fig. 2, P < 0.05). In relation to the bacterial community, noticeable temporal shifts were identified, demonstrating a substantial distinction between the control group (CK) and the sulfur-treated group at the initial stages of incubation (days 14, 30, and 60). These findings underscore the significant impact of sulfur on the beta diversity of the bacterial community over time. Conversely, the fungal community displayed a distinct pattern. While sulfur treatment exerted minimal influence on the beta diversity of the fungal community, there was a distinctive clustering pattern apparent only among the samples collected on day 60. This suggests that the fungal community’s response to sulfur treatment might be more nuanced and time-dependent, with specific clustering only becoming apparent after an extended period of exposure. The observed stability in alpha diversity, despite temporal changes in beta diversity, may be explained by the fact that alpha diversity reflects the overall species richness and evenness within individual samples, which remained relatively constant over time. These findings align with previous studies highlighting that minor shifts in key microbial groups can still occur without substantially altering the total diversity metrics [22, 23]. In contrast, beta diversity reflects the variation in microbial composition across different samples and time points. These temporal shifts in beta diversity suggest changes in the relative abundances of specific microbial groups in response to sulfur treatment, even though the overall microbial richness remained stable. These results highlight the complex interplay between sulfur treatment, microbial communities, and temporal dynamics, shedding light on the intricate ecological responses within the rubber plantation ecosystem.

Fig. 2

PCoA of the Bray-Curtis distance between bacterial (a) and fungal communities exposed to sulfur during the 60-day incubation. The Adonis analysis reveals significant beta diversity among the samples (P < 0.05), indicating differences in community composition

Microbial composition and sulfur perturbation in rubber plantation soils

The high-throughput sequencing analysis uncovered the presence of around 28 bacterial and 11 fungal phyla across all soil samples, shedding light on the diverse microbial landscape within the rubber plantation ecosystem. Such diversity is consistent with previous findings in tropical agroecosystems [24, 25]. The intricate composition of both bacterial and fungal communities is illustrated in Fig. 3a-b, accentuating the prevalence of specific phyla with relative abundances exceeding 1%. Notably, the most abundant bacterial phyla, including Acidobacteriota, Actinobacteriota, Chloroflexi, Proteobacteria, and Firmicutes, collectively constituted more than 80% of the total sequences in all samples (Fig. 3a; Table S2). Of particular interest, the Chloroflexi phylum plays a key role in dechlorinating and detoxifying various anthropogenic contaminants [26], highlighting its ecological importance in maintaining soil health. However, the presence of sulfur and its compounds, such as sulfite and sulfide, appeared to disrupt the stability of this community, impairing the reductive dechlorination activity of Chloroflexi species responsible for organohalide degradation [27]. The findings indicate that the relative abundance of Chloroflexi initially rose in the sulfur treatment on days 14 and 30, followed by a sharp decline by day 60 compared to CK. This transient disruption could have long-term ecological implications, as Chloroflexi plays a crucial role in the degradation of organohalides, compounds commonly found in contaminated soils [28]. A decline in Chloroflexi abundance may reduce the soil’s capacity to degrade harmful organohalides, potentially slowing down the detoxification process over time. Furthermore, the relative abundance of Planctomycetes was found to be significantly lower in the sulfur treatment compared to CK on days 14 and 30, suggesting that sulfur exposure could influence the dynamics of this particular bacterial group.

Fig. 3

Impact of sulfur on bacterial (a) and fungal (b) community composition at the phylum level

Within the fungal community of the rubber plantation soil, the dominant presence of Ascomycota and Basidiomycota stood out, accounting for a substantial proportion of the total sequences, ranging from 79.7 to 88.2% (Table S3). These findings align well with previous research studies, corroborating the consistent prevalence of these fungal phyla in similar ecosystems. Furthermore, the relative abundance of Mortierellomycota in the sulfur treatment remained consistently below 1% [29, 30]. This observation implies that sulfur treatment might have a suppressing effect on the abundance of Mortierellomycota, possibly attributed to the acidification of the soil driven by sulfur application [11, 31]. Mortierellomycota are known to play important roles in soil nutrient cycling and organic matter decomposition, particularly in symbiotic relationships with plants and other soil organisms [32, 33]. The suppression of this fungal group due to soil acidification could have broader implications for soil health, as it may reduce the efficiency of nutrient cycling and organic matter breakdown. In the long term, such changes could disrupt soil ecosystem functions, leading to altered nutrient availability and potentially affecting plant growth and soil stability.

At the genus level (Fig. S2), sulfur exposure correlated with an increased relative abundance of Bacillus, Sinomonas, Ktedonobacteraceae 1921-2, and Catenulispora. Notably, Bacillus is capable of utilizing sulfide to form cysteine and has been recognized for its ability to remove sulfur from organosulfur compounds [34,35,36]. Similarly, two strains of Sinomonas have demonstrated the capability to remove over 40% of sulfur from coal [37, 38], suggesting that these bacteria might have harnessed sulfur elements to facilitate their own reproduction. Moreover, the LEfSe analysis highlighted the sensitivity of six bacterial biomarkers to sulfur treatment (p < 0.05, LDA > 2.0; Fig. 4). Notably, the sulfur treatment exhibited an increased relative abundance of specific taxa, including Chloroflexi TK10 from days 7 to 30, Ktedonobacteraceae FCPS473 from days 14 to 30, and Bryobacter, Diplorickettsiaceae, Clostridium_sensu_stricto_8, and Thermomicrobiales on days 7 and 30. These bacterial species play significant functional roles in soil nutrient cycling processes. For example, Chloroflexi TK10 is a pivotal bacterium that influences the dissolved organic matter in forest soils during nitrogen deposition [39]. Bryobacter is an aerobic chemoheterotroph involved in carbon cycling and is considered a core and keystone genus within forest ecosystems [40]. Additionally, Thermomicrobiales have the potential to contribute to soil CO₂ emissions by oxidizing CO [41]. The higher abundance of these bacteria in the sulfur treatment, particularly within days 30, suggests that sulfur treatment exerts a short-term promoting effect on soil nutrient cycling.

Fig. 4

Identification of differentially abundant bacteria between the CK and sulfur treatment based on the linear discriminant analysis effect size analysis (LEfSe). The score was utilized to quantify the degree of differentiation between CK and sulfur, employing a threshold value of 2.0

Sulfur-induced alterations in microbial interaction networks and keystone taxa in rubber plantation soils

The modifications in the configuration of co-occurrence networks and the presence of keystone taxa may serve as indicators of the influence of pesticides on the stability and potential functions of soil microbial communities [42, 43]. In the context of this study, an ecological network analysis was conducted to examine the effects of sulfur on interactions between bacterial and fungal genera within the same species. The co-occurrence networks of bacteria and fungi, along with their topological characteristics under both CK and sulfur-treated conditions, are depicted in Fig. 5. Notably, the number of connections within the bacterial co-occurrence network increased from 436 in CK to 482 in the sulfur-treated group. Furthermore, the proportion of positive correlations rose from 60.32% in CK to 64.94% in the sulfur-treated group. The greater graph density and average degree observed in the sulfur-treated group highlight the formation of a more intricate and closely connected bacterial network compared to CK. These findings imply that exposure to sulfur enhances bacterial interactions, particularly in response to challenging environmental conditions [44, 45]. In particular, the co-occurrence network of the sulfur-treated group displayed a distinct cluster of robust correlations among Acidobacteria species, as illustrated in Fig. 5a. Environmental factors such as pH and nutrient availability have been observed to influence the dynamics of Acidobacteriota populations [27]. Many Acidobacteriota species are known to thrive in acidic conditions and exhibit a higher abundance under such circumstances [46]. The findings from this study offer further support for the utilization of sulfur as a means of plant protection, specifically in lowering soil pH [11].

Fig. 5

Effects of sulfur on bacterial (a) and fungal (b) co-occurrence networks. The distinct bacterial and fungal taxa at the phylum level are represented by different colors. The magnitude of each node is commensurate with its degree. A link signifies a robust correlation (Spearman’s r > 0.8) with statistical significance (p-value < 0.01). Positive correlations are depicted by red edges, while negative interactions between two nodes are indicated by blue edges

As for the fungal co-occurrence networks, there was a reduction in the number of connections (200 vs. 287) and average degree (4.878 vs. 6.753) in the sulfur-treated group compared to CK. Interestingly, the sulfur-treated group exhibited an increase in positive correlations (62.00% vs. 59.93%) compared to CK. This observation suggests that the fungal community also engages in intraspecific cooperation in response to sulfur influence. Additionally, networks were established to investigate the influence of sulfur on bacterial-fungal interactions (Fig. 6). The number of connections between different species decreased from 476 in CK to 296 in the sulfur-treated group. Simultaneously, positive correlations between different species increased from 56.72 to 62.84%. These results indicate that sulfur treatment leads to a reduction in pivotal species within the bacteria-fungi network while fostering interspecific cooperation within microbial communities. However, it’s important to note that excessive cooperation could potentially amplify the spread of environmental disruptions across the community, compromising overall community stability [47]. Such network-level changes underscore the need for cautious sulfur application to maintain functional balance in rubber plantation soils. Consequently, the changes in microbial topological roles brought about by sulfur within these networks could foster excessive cooperation among minority microorganisms, both intraspecifically and interspecifically. This, in turn, might compromise community stability and therefore, impact soil biogeochemical cycles within rubber plantations.

Fig. 6

Effects of sulfur on the bacterial-fungal co-occurrence network. Positive correlations are depicted by red edges, while negative interactions between two nodes are indicated by green edges

Impacts of sulfur spraying on microbiome phenotypes and functional resilience in rubber plantation soils

Diverse microbial species within soil ecosystems exhibit distinct ecological functions. In this study, phenotypic changes within the soil bacterial community were assessed using BugBase (Fig. 7a). A single species can exhibit multiple phenotypes, resulting in a cumulative phenotypic abundance that often exceeds 100% within a group. In the surface soil of rubber plantations, the abundance of aerobic bacteria was significantly higher than that of anaerobic bacteria, and Gram-negative bacteria were more abundant than Gram-positive bacteria, mirroring trends reported in similar agroecosystems [48]. Both PICRUSt2 and Tax4Fun analyses revealed the variety of functional categories within the soil bacterial communities of rubber plantations (Fig. 7b, c). Throughout the rubber plantation, the microbial phenotypic abundances and functional categories within the bacterial community remained relatively stable, showing minimal impact from sulfur treatment. These findings align with the crucial role that diverse and abundant soil microorganisms play in the decomposition and nutrient cycling processes within ecosystems [49]. To delve deeper, the FAPROTAX methodology was utilized, which relies on the functional annotation of the prokaryotic microbial taxonomic database. This approach allowed to anticipate the possible impacts of sulfur on soil functions associated with the cycling of carbon, nitrogen, and sulfur. As illustrated in Fig. 7d, the addition of sulfur notably inhibited the respiration intensity of sulfur compounds. This suggests that sulfur treatment had a disruptive effect on the metabolic activity of sulfate-reducing bacteria within the soil, potentially compromising soil sulfur metabolism. Overall, these results underscore the intricate relationship between soil microorganisms, their functional roles, and the potential impacts of sulfur treatment on vital biogeochemical processes.

Fig. 7

Changes in bacterial function annotated by BugBase (a), PICRUSt2 (b), Tax4Fun (c), and FAPROTAX (d) databases under sulfur stress

The temporal shifts observed in microbial communities due to sulfur application are significant, but it is important to note that these findings are based on a 60-day study period. Further research is necessary to understand the long-term effects of sulfur treatment on soil microbial communities. Although sulfur application significantly influenced the relative abundance of specific taxa, its broader ecological implications and exact mechanisms warrant additional study, particularly under longer-term or higher-intensity sulfur treatments. The results should be interpreted in the context of rubber plantations with similar soil and environmental conditions, and may not be directly applicable to all agricultural settings. In addition, future studies should include additional independent replicates and investigate multiple soil types under rubber cultivation to strengthen the evidence base on sulfur’s effects. This broader approach would enable a comprehensive assessment of how soil texture, pH, nutrient profiles, and other properties interact with shifts in microbial communities under long-term sulfur application. In particular, developing a more detailed correlation between microbial community composition and key soil parameters (e.g., carbon, nitrogen, and sulfur availability) will help clarify the ecological processes governing microbial succession in response to sulfur inputs.

Conclusion

This study uncovers the effects of sulfur application on soil microbial communities and functional attributes in rubber plantation soil. The application of 45 kg of sulfur per hectare led to temporal shifts in microbial community structures and influenced the composition of specific bacterial and fungal taxa over a 60-day period. Certain bacteria (e.g. Bacillus and Sinomonas) flourished under sulfur treatment, influencing nutrient cycling. While sulfur spraying enhanced microbial interactions and cooperation, it also had a limited impact on microbial phenotypes and community functions. Additionally, sulfur application inhibited the respiration of sulfur compounds, indicating a more complex effect on soil microbial activity. These findings highlight that elemental sulfur can have both beneficial and adverse effects on soil microbial dynamics in rubber plantations. This emphasizes the need for carefully balancing sulfur application to maintain soil health and prevent negative impacts on long-term soil fertility.