Rust Disease Changes the Abundance and Composition of Bacterial Community in Iris lactea Rhizosphere

Abstract

The microbial community plays a vital role in root–environment interactions, which affect plant performance under biotic stress. Rust disease significantly affects plant growth, which may also affect rhizosphere microbial community. However, there is a scarcity of studies investigating the microbial community of rhizosphere under rust disease stress. Iris lactea is a widely utilized plant in gardening and landscaping due to its versatility and ornamental value, but it is often susceptible to rust disease in landscape settings. In this study, we compared the bacterial communities between bulk soil (non-cultivated control), rhizosphere soil of healthy Iris lactea plants, and rhizosphere soil of Iris lactea plants infected with rust disease (rhizosphere-R). Results revealed significant alterations in the abundance and composition of bacterial communities associated with rust disease infection. Specifically, the rhizosphere-R samples exhibited a decreased Shannon index at 1.9% compared to bulk soil and the relative abundance of Proteobacteria was increased at 31.65%. Moreover, distinct changes in β-diversity were shown between bulk soil and rhizosphere samples. Notably, potentially pathogenic bacteria increased in abundance under rust disease stress, while beneficial bacterial taxa decreased. Overall, our results show that rust disease affects the rhizosphere microbial community, which emphasizes the ecological implications of plant–microbe interactions under biotic stress and implications for developing targeted rhizobacterial-based biocontrol strategies.

1. Introduction

The rhizosphere harbors complex and diverse microbial communities regulated by plant root exudates, and this narrow region is also a crucial hotpot area in plant–microbe interactions [1,2]. The microorganisms located in the rhizosphere play vital roles in plant health, nutrient cycling, and stress resistance [3]. Among these microorganisms, rhizobacteria are particularly significant due to their direct involvement in promoting plant growth and suppressing soil-borne pathogens [4]. Several studies focusing on plant disease–microbiome interactions indicated that infections with pathogens aboveground alter the plant’s rhizosphere microbial community [5,6]. The rhizosphere microbial community may also act as the source of the phyllosphere microbial community, which determine the aboveground productivity and plant health [7,8]. Until now, most studies about phyllosphere disease have focused on the phyllosphere microbiome, but systemic research of its effect on the rhizosphere microbial community under pathogen invasion aboveground remains inconclusive. Due to the microbial community assembly of rhizosphere being largely governed by the whole plant growth environment [9,10], understanding the composition and function of rhizobacterial communities is crucial for developing sustainable agricultural and horticultural practices, especially in the context of disease management.

Iris lactea var. chinensis is an ornamental plant widely used in gardening for its aesthetic value and adaptability to various environmental conditions. However, this plant is not immune to diseases, with rust disease being one of the major biotic stresses affecting the foliage. Rust diseases, caused by fungal pathogens, can severely affect plant growth and ultimately lead to yield losses in agricultural settings [11,12]. Despite the significance of Iris lactea var. chinensis in horticulture, the impact of rust disease on its rhizobacterial community remains largely unexplored. It is essential for plants to deal with environmental signals, especially the biotic and abiotic stresses [13]. The specific assembly of soil microbial communities in rhizosphere is affected by a variety of biotic and abiotic factors [14]. Each plant species harbors a specific rhizosphere community [15], but few studies have been conducted on the impacts of aboveground biotic stresses on rhizosphere microbial communities. Studies have shown that aboveground diseases can affect rhizosphere microbial communities and foliar application with PGPR can also alter the microbial composition of pine rhizosphere [16]. Another fungi pathogen Botrytis cinerea caused leaf disease and also changed the bacterial interactions in the root–substrate interface [17]. Furthermore, the changes in root exudates induced by biotic stress composition alter the rhizospheric microbial community and, thus, confer plant stress tolerance [18].

Recent advances in sequencing technologies have enabled detailed characterization of microbial communities associated with plant roots, offering unprecedented insights into the structure and function of rhizobacterial assemblages [19]. The assessment of microbial diversity is crucial for our understanding of the functionality and stability of plant–environment interactions [20,21]. The alpha-diversity is often measured as the observed richness (number of taxa) or evenness (the relative abundances of those taxa) of an average sample within a habitat type [22]. The beta-diversity is the variability in the identity of taxa observed among samples within a habitat, including the rhizosphere [23]. By comparing the rhizobacterial communities of healthy and rust-diseased Iris lactea plants, this study aims to elucidate the potential shifts in bacterial community composition and diversity that may be associated with rust disease infection. Specifically, we hypothesize that rust disease alters the rhizobacterial community structure, potentially disrupting beneficial plant–microbe interactions and favoring opportunistic or pathogenic microorganisms.

In this study, we investigated the impact of rust disease on the rhizosphere microbial community of Iris lactea var. chinensis. We analyzed bacterial communities in three distinct soil types: the soil near the sampling sites that is not cultivated with Iris lactea var. chinensis (Bulk soil), rhizosphere soil of healthy Iris lactea plants (rhizosphere), and rhizosphere soil of Iris lactea plants infected with rust disease (rhizosphere-R). By comparing these microbial communities, we aimed to elucidate potential shifts in bacterial diversity and composition associated with rust disease infection.

Our hypothesis is that rust disease alters the rhizobacterial community structure, potentially disrupting beneficial plant–microbe interactions and favoring opportunistic or pathogenic microorganisms. This study aims to contribute to the existing knowledge on plant–microbe interactions under disease stress and could pave the way for developing targeted rhizobacterial-based biocontrol strategies for managing rust diseases in ornamental plants. Furthermore, our findings may provide insights into the broader ecological implications of plant–microbe interactions under disease pressure, emphasizing the importance of considering both above- and below-ground dynamics in disease management.

2. Materials and Methods

2.1. Sampling Sites and Samples of Iris Lactea

The Iris lactea var. chinensis were collected from a garden nursery in Jinan City, Shandong Province, where the important garden plant Iris lactea var. chinensis is cultivated. The Iris lactea plants were transplanted at the same time and grown in the same environment. The sampling sites are located at 36°83′ N latitude, 117°32′ E longitude. The soil was classified as sandy loam, and the 0–20 cm layer soils had a bulk density of 1.42 g cm⁻³, a pH of 8.04, and an electrical conductivity (EC) of 0.41 mS cm⁻¹. The content of soil organic C is 6.5 g kg⁻¹ and of soil total nitrogen (N) is 0.45 g kg⁻¹. For the quick-acting soil nutrient content, the soil contained 42.5 mg kg⁻¹ mineral N (nitrate N plus ammonium N), 17.5 mg kg⁻¹ of available phosphorus (P), and 130.5 mg kg⁻¹ of available potassium (K). Both the rhizosphere soil samples of Iris lactea var. chinensis and the bulk soil samples used in this study were collected from the same soil characteristic region. The bulk soil and the Rhizosphere soil of plants were sampled using the procedure as described in Niu et al. [24]. Briefly, the Iris lacteal plants were carefully harvested and the large soil aggregates around plant were removed by shaking the roots. Then, the roots were put into a 50 mL tube containing 25 mL sterile 1 × PBS buffer (180 rpm, 20 min). After that, the roots were removed and the PBS buffer was subjected to centrifugation (10,000× g, 10 min). Moreover, the PBS buffer was removed and soils in tube were defined as the rhizosphere soil compartment.

2.2. Analysis of Amplicon Sequencing Data

For analyzing the diversity of bacterial community, genomic DNA of different samples was extracted and purified from about 400 mg soil using a PowerSoil DNA Isolation Kit (Qiagen, Valencia, CA, USA). To target the V3-V4 region in 16S rRNA gene, we used primer F (5′-ACTCCTACGGGAGGCAGCA-3′) and reverse primer R (5′-GGACTACHVGGGTWTCTAAT-3′). The PCR amplification was performed under the following conditions: 95 °C for 3 min; followed by 27 cycles of 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 45 s; and a final step at 72 °C for 10 min. PCR products were purified using the QIAquick PCR purification kit (Qiagen, Valencia, CA, USA) when no visible amplification was observed from negative control and subjected to a single sequencing run on the MiSeq platform (Illumina, San Diego, CA, USA).

2.3. Statistical Analysis

For analyzing the soil bacterial community, rhizosphere soil-based Illumina sequences of 16S rRNA were processed and sequentially quality-filtered using Trimmomatic (v0.33). The operational taxonomic units (OTUs) of different samples were clustered using a 97% similarity cut-off via the Ribosomal Database Project’s classifier [25] and the SILVA database (version 138) [26] for bacteria. Then, the OTUs identified as belonging to chloroplast and mitochondria were removed from the data set. Next, the representative sequences for each OTU were aligned using PyNAST in QIIME2 [27]. To eliminate the potential bias of different soil samples caused by different sequence depth, we used the minimum number across all samples to keep all samples at the same depth. Several indexes including Shannon index, Chao1, and Simpson index were applied to represent the α-diversity of the different soil bacterial communities. PCoA based on unweighted UniFrac distance was used to further analyze the β-diversity of bacterial composition. Linear discriminant analysis (LDA) of effect size (LEfSe analysis) was applied on the OTU level to identify the differentially abundant bacterial taxa (at genus to phylum levels) that significantly change between bulk soil and rhizosphere soil. Wilcoxon rank-sum test for pairwise comparison (false discovery rate (FDR) adjusted p < 0.05) and the absolute LDA score (>4) were used to analyze the statistical significance and strength, respectively. The relative indicators were analyzed on the online platform of BMKCloud (https://www.biocloud.net/, accessed on 4 January 2024). The raw sequencing data of the bacteria were submitted into the NCBI Sequence Read Archive (SRA) database (Accession Number: PRJNA1152242).

3. Results

3.1. The Phenotype of Rust Disease and Raw Data Quality Control of Bacterial Community

We present two contrasting phenotypic expressions of leaf rust disease in Iris lactea var. chinensis, specifically focusing on the leaves of the plant (Figure 1). The left panel illustrates a severe phenotype, characterized by the presence of numerous, elongated, and distorted brown lesions that are densely packed on the leaf surface (Figure 1a). In our study, we employed 16S rRNA sequencing to characterize the bacterial community and adopted an absolute quantitative analysis approach for data interpretation. In our study, we employed 16S rRNA sequencing to characterize the bacterial community to determine the response to rust disease. All rarefaction curves for bulk soil and rhizosphere bacteria community analysis tended to plateau, indicating that the sequencing effort was sufficient for the overall bacterial community diversity (Figure 2). After double-end sequence data were spliced and filtered, a total of 346,286 bacterial sequences and 7191 OTU numbers were detected. The number of effective sequences of bacteria was 24,158–45,354, respectively, in different samples. In total, 7191 OTUs of bacteria were detected in all soil samples, belonging to 36 phylum, 89 classes, 238 orders, 443 families, 778 genera, and 911 species (Table S1).

3.2. Microbial α-Diversity and Rarefaction Curve

The presented figure comprehensively compares the α-diversity and rarefaction curve of microbial communities within the bulk soil, rhizosphere, and rhizosphere-R samples of Iris lactea var. chinensis. The rarefaction curve exhibits an initial steep rise followed by stabilization, confirming the successful amplification of the 16S rRNA gene, a crucial step in ensuring the reliability of subsequent diversity analyses (Figure 2).

The box plots in Figure 2 reveal distinct patterns in microbial diversity among the sample groups. Specifically, the bulk soil exhibits higher Shannon diversity indices based on observed operational taxonomic units (OTUs) (Figure 2b) compared to the rhizosphere and rhizosphere-R samples. This finding underscores the greater microbial diversity present in the bulk soil, potentially reflecting a less selective environment compared to the rhizosphere and rhizosphere-R, where microbial communities may be influenced by plant-specific factors.

The rhizosphere and rhizosphere-R samples, on the other hand, do not differ significantly in their Shannon indices or observed OTUs, suggesting a similar level of microbial selectivity within these two compartments. But the mean values of both diversity indices for the bulk soil consistently exceed those of the rhizosphere and rhizosphere-R. Specifically, the rhizosphere-R samples exhibited a decreased Shannon index at 1.9% compared to bulk soil.

3.3. Composition of Bacterial Communities in the Rhizosphere

The figure reveals notable changes in the bacterial community composition across these soil types. The relative abundance of Proteobacteria in rhizosphere-R samples was increased compared to bulk soil, at 31.65%. Specifically, the rhizosphere of healthy Iris lactea var. chinensis plants exhibits a distinct bacterial community profile compared to bulk soil, while the rhizosphere of plants under rust disease stress shows further alterations in bacterial species abundance and composition (Figure 3). At phylum level, the relative abundance of Acidobacteriota in the rhizosphere soil of Iris lactea decreased compared to bulk soil, while the relative abundance of Proteobacteria increased (Figure 3a). Compared to the bulk soil, the relative abundance of phylum Actinobacteria was decreased, especially in the Iris lactea rhizosphere under rust disease stress. The genus RB41 was decreased in both Iris lactea rhizosphere soils. Similarly, Sphingomonas also exhibits the same decreasing trend. Surprisingly, in the rhizosphere of Iris lactea var. chinensis, the relative abundance of the genus Allorhizobium increased significantly.

3.4. Rust Disease Alters the β-Diversity of Rhizosphere Soil

Rust disease could not significantly alter the α-diversity of Iris lacteal rhizosphere bacterial community (Figure 3); however, it strongly affected the β-diversity of different bacterial communities. PCoA analysis based on unweighted unifrac distances was applied to determine the overall bacterial community structure of rhizosphere soil and showed significant separation between different soil samples (Figure 4a), indicating significant differences in their microbial compositions. Notably, the rhizosphere and rhizosphere-R communities cluster closely, suggesting a higher degree of similarity compared to the bulk soil community.

The PCoA plot showing the first principal coordinate (PC1), which explains 18.14% of the total variation, further substantiates these findings. The bulk soil samples occupy a distinct region on the left side of the plot, whereas the rhizosphere and rhizosphere-R samples are positioned on the right, indicating their distinct microbial profiles. The box plot accompanying the PCoA plot illustrates the statistical distribution of PC1 values across the groups. The bulk soil group exhibits a narrower range and median value distinct from those of the rhizosphere and rhizosphere-R groups, which show more overlap and a wider spread of PC1 values.

The PERMANOVA analysis shows that the statistical significance of the PCoA model is underscored by the R² value of 0.297 and a highly significant p-value of 0.001, indicating that the observed differences among the groups are not merely random but rather reflect genuine biological variations (Figure 4b). The box plots highlight the distinctiveness of the rhizosphere-R community, as evidenced by its more extreme PC1 values compared to the rhizosphere and bulk soil groups.

Furthermore, the LefSe analysis offers a nuanced understanding of microbial composition and relative abundances at various taxonomic levels, from kingdom to species (FDR-adjusted p < 0.05, Wilcoxon rank-sum test, and absolute LDA score > 4). The orders Vicinamibacterales and Saccharimonadales were enriched in the rhizosphere of Iris lactea (Figure 5), and the class Vicinamibacteria was also enriched (Figure 5).

In the bulk soil samples, p_Proteobacteria emerges as the dominant phylum, exhibiting the highest LDA score, signifying its substantial abundance. This is followed by e_Gammaproteobacteria and o_Burkholderiales, which also contribute significantly to the microbial community. Conversely, o_Sphingomonadales displays the lowest LDA score, indicating its relatively low abundance in bulk soil. Transitioning to the rhizosphere samples, notable shifts in bacterial abundance become evident. Although p_Proteobacteria maintains its dominance, its LDA score decreases, suggesting a decrease in abundance compared to that of bulk soil. Conversely, f_Rhizobiaceae and g_Alkalihalobacterum_Nostocohabans_Paraburkholderium_Rhizobium exhibit increased abundance, as reflected by their higher LDA scores. Additionally, s_unclassified_Alkalihalobacterum_Jium_Paraburkholderium_Rhizobium and p_Bacteroidetes also show enhanced presence in the rhizosphere, highlighting the unique microbial composition of this soil environment. Further analysis of the rhizosphere-R samples reveals further alterations in bacterial abundance patterns. p_Proteobacteria regains its dominance, with e_Gammaproteobacteria and o_Burkholderiales also displaying increased LDA scores. Notably, f_Sphingomonadaceae and g_unclassified_Sphingomonadaceae become more abundant, whereas e_Sphingomonadales and g_unclassified_Sphingomonadaceae exhibit a decrease in abundance. These findings underscore the dynamic nature of microbial communities in response to different soil conditions and treatments. Collectively, the results presented in this figure underscore the distinct bacterial community compositions observed in bulk soil, rhizosphere, and rhizosphere-R samples.

3.5. BugBase Phenotype Prediction of Rhizosphere Bacterial Community

We employed BugBase, which is a novel method for analyzing complex microbiome data to determine biologically relevant microbiome phenotype predictions at organism level. Firstly, the relative abundance of potentially pathogenic bacteria in different samples was shown. This analysis reveals a notable variation in bacterial abundance among the soil types, with rhizosphere-R samples exhibiting the highest relative abundance of the potentially pathogenic trait. This observation underscores the significance of the rhizosphere soil environments in harboring potentially pathogenic bacteria under rust disease stress. Furthermore, a bar plot also categorizes the relative abundance of the trait of stress tolerance in bulk soil and rhizosphere-R samples. The results indicate a clear trend: bacteria with high stress tolerance dominate both soil types, suggesting their enhanced adaptability and resilience under adverse conditions (Figure 6).

To delve deeper into taxonomic composition, a stacked bar plot illustrates the relative abundance of specific bacterial groups associated with the trait potentially pathogenic, including Comamonadaceae, Xanthomonadaceae, and Rhizobiaceae, in rhizosphere and rhizosphere-R samples. Under rust disease stress, the relative abundance of Xanthomonadaceae and Comamonadaceae was increased, highlighting their ecological significance in these environments. Additionally, a more intricate stacked bar plot combines stress tolerance categories with taxonomic groups to provide a holistic view of bacterial distribution. This analysis confirms that bacteria with high stress tolerance, such as Shinyegpseyae and Comamonadaceae, are more abundant in both bulk soil and rhizosphere-R samples compared to their low-stress-tolerance counterparts (Figure 6d).

4. Discussion

The rhizosphere microbial community serves as a crucial biological regulator for plant stress resistance responses [1,28,29]. The plant bacterial community is diverse and contributes to different aspects of plant health, especially under biotic and abiotic stresses [30,31]. Our results demonstrate a significant impact of rust disease on the rhizosphere microbial community of Iris lactea var. chinensis. The distinct microbial patterns observed in the bulk soil, rhizosphere of healthy plants, and rhizosphere of rust-infected plants offer valuable insights into the complex interplay between plant health and rhizosphere microbial ecology. The rhizosphere-R samples, representing the plant under rust disease, exhibited notable alterations in bacterial community composition and structure compared to their healthy counterparts, suggesting that rust disease not only affects plant health but also triggers profound shifts in the microbial environment of the rhizosphere. The Shannon diversity indices were decreased compared to those of the bulk soil, showing that the roots favor certain microbial populations; this is consistent with the result that the diversity of rhizosphere soil bacteria decreased after southern root-knot nematodes infected cucumber roots [32]. Plants can recruit various soil-beneficial microorganisms into the rhizosphere when dealing with specific environmental stresses, including biotic stress aboveground [28]. Further, in the related research, disease-induced recruitment of soil-beneficial bacteria induced systemic resistance against downy mildew disease and eliminated the growth disadvantage [5,33]. Several studies have reported similar findings in other plant–microbe systems. Microbial community establishment in the rhizosphere is driven by plant root exudates, which could be affected by biotic stress [34,35], and pathogen infection can specifically accumulate a group of beneficial microbes, which may lead to the decrease in α-diversity [34]. Moreover, Bardgett and van der Putten emphasized the critical role of belowground biodiversity in ecosystem functioning, highlighting how plant–microbe interactions can be disrupted by disease stress [15]. Similar to our observations, Mendes et al. found that rhizosphere microorganisms play crucial roles in plant health, nutrient cycling, and stress resistance [36]. Our results align with these studies, underscoring the delicate balance between plant health and rhizosphere microbial communities.

Under the stress of rust disease, the bacterial community diversity of different soil samples did not undergo significant changes, but the bacterial community structure and composition were notably altered, suggesting potential alterations in root exudates. Recent research indicated that host selection could cause a greater determining effect on shaping the plant microbiome than the environmental factors [37]. The increase in the relative abundance of potentially pathogenic bacteria in rhizosphere-R samples, as predicted by BugBase analysis, is consistent with findings from previous research, which reported shifts towards more opportunistic or pathogenic microbial taxa under stress conditions [38]. The family of Xanthomonadaceae was enriched in the bacterial community of Iris lactea rhizosphere infected by rust disease. Numerous pathogens exist within the family of Xanthomonadaceae; therefore, infection in the aboveground parts may also lead to the recruitment of more pathogens by the root system, further affecting plant growth [39]. The family of Comamonadaceae was also enriched compared to bulk soil. However, in the stress tolerance traits, the relative abundance of the Comamonadaceae and Xanthomonadaceae families was increased, showing the specific recruitment in Iris lactea rhizosphere. There are also studies showing that the helpful microorganisms, such as Comamonadaceae and Xanthomonadaceae, were enriched in Fusarium-vulnerable cucumber plants to inhibit the Fusarium pathogen by producing higher levels of organic acids [40]. These results demonstrate that rust disease could also affect the specific bacterial taxa in the Iris lactea rhizosphere. This indicates that the bacterial community shaped by Iris lactea roots under the influence of rust disease is dual-faceted, harboring both pathogenic microorganisms and enriching beneficial bacteria.

Furthermore, the observed decrease in microbial diversity in rhizosphere-R samples, despite insignificant changes in α-diversity indices, is in line with studies showing that microbial community structure can be more sensitive to environmental perturbations than overall diversity. This indicates that rust disease primarily affects the composition rather than the overall richness of microbial communities in the rhizosphere.

5. Conclusions

In conclusion, our study demonstrates that rust disease significantly alters the abundance and composition of the rhizosphere bacterial community of Iris lactea. This observation underscores the importance of considering both above- and below-ground plant–microbe interactions when studying plant disease resistance and developing effective biocontrol strategies. Our findings have important scientific value for advancing our understanding of plant–microbe interactions under disease stress and provide a foundation for future research aimed at exploiting the rhizosphere microbiome for sustainable plant health management. However, how rust disease affects the bacterial communities in the rhizosphere is still unknown and needs further in-depth studies. Future research should prioritize the investigation of the changes in rust-induced root exudates and their significance in shaping the rhizosphere bacterial community.