Characteristics of rhizosphere and bulk soil microbial communities in pear trees

Introduction

In the process of evolution, different plants have developed various defense mechanisms to cope with different environmental stresses, such as temperature, salinity, drought, pests and pathogens. The rhizosphere of plant, a special ecological niche on plant, has gradually attracted attention in recent years due to the diversity functions of the bacterial communities colonizing it. The rhizosphere was initially delineated as a zone of heightened bacterial activity in the soil adjacent to the plant roots (Lynch & Whipps, 1990) that has different interactions with plants. The interaction modes between different plants and rhizosphere bacteria can be divided into two main categories. Some rhizosphere bacteria are beneficial bacteria for plants (Berendsen et al., 2018). They can promote plant growth, antagonize and compete with pathogens and induce plant resistance, such as Bacillus (Xia et al., 2024), Pseudomonas (Susanne et al., 2018), Arthrobacter (Xu et al., 2021). Some bacteria are potential soil-borne pathogens, leading to plant yield reduction and so on (Bains et al., 2009). Blom et al. (2011) tested the effects of volatiles produced by 42 strains of plant rhizosphere bacteria on plant growth, and found that in addition to significantly increasing plant biomass, these volatiles could also lead to plant death. Xu et al. (2021) found that the relative abundance of bacteria with potential pathogenic phenotypes was higher in the rhizosphere of peanut. Therefore, plants need to balance their rhizosphere microbial ecosystem through the interaction with different bacteria.

The rhizosphere region harbors a large number of microorganisms that have different biological functions in the progress of the interactions with host plants (Xu et al., 2021; Egamberdieva et al., 2008). The rhizospheres of many plants, such as wheat, bean, kiwifruit and banana selected beneficial microorganisms from the bulk soil to address biotic and abiotic stresses (Ling, Wang & Kuzyakov, 2022; Jamil et al., 2022; Cardoso et al., 2021; Yin et al., 2021; Yan et al., 2023). Within the rhizosphere of Capsicum annuum, a microbiome with the ability of promoting plant photosynthetic performance and biomass synthesis was enriched to mitigate drought effects (Rodriguez et al., 2008). Some microbiome with the ability of promoting plant growth and nutrient assimilation were enriched in sorghum and peanut rhizospheres (Xu et al., 2021; Alsabri et al., 2020). Yin et al. (2021) found that successive wheat plantings and application of pathogen could specifically accumulate a group of beneficial microbes with antagonistic activities. Yan et al. (2023) isolated a large number of antagonistic bacteria from the kiwifruit rhizosphere, such as Bacillus and Acinetobacter, which have antagonistic effects against Pseudomonas syringae pv. actinidiae. Enriched Flavobacterium in the rhizosphere of resistant tomato plants could effectively suppress the occurrence of Ralstonia solanacearum in susceptible plants (Kwak et al., 2018). In addition, Ling, Wang & Kuzyakov (2022) analyzed the microbial composition and functions of the rhizosphere soil and the bulk soil from different ecosystems, including farmland crops, herbaceous plants, forest trees and the main differences were generalized. The rhizosphere selected some microorganisms from the bulk soil to function as a seed bank, but the bacterial diversity was reduced by 0.9%−5.3%. Compared with the bulk soil, the abundances of Bacteroidetes, Proteobacteria and Actinobacteria increased, whereas those of Nitrospirae, Gemmatimonadetes, Acidobacteria and Chloroflexi decreased in the rhizosphere soil. All bacterial metabolic and ecologically relevant functional predictions showed that genes involved organic compound conversion, nitrogen fixation, denitrification and pathogenesis were enriched in rhizosphere soil, while genes involved in nitrification were decreased. As the rhizosphere was a special ecological zone connecting plant roots and soil and played a crucial role in the dynamics of soil C and N dynamics, it hosted diverse bacteria capable of performing C and N transformation-related functions (Ling, Wang & Kuzyakov, 2022). As an indispensable fruit in daily life, it remains unknown whether the rhizosphere of pear trees can enrich certain beneficial microorganisms when growing naturally.

Pears are one of the most important fruits in the world and are widely cultivated in many countries, including China, the United States, Spain etc. In recent years, as people’s living quality have risen, the demand for high-quality pears has steadily increased. To fulfill the expectations of consumers, some excellent pear tree varieties have emerged (Wu et al., 2013; Zhang et al., 2010). In particular, ‘Yuluxiang’ is a high-quality pear tree variety with high-sugar-content, crisp and juicy. It is primarily cultivated in the northern part of China. However, in recent years, the spread of various fruit tree diseases has seriously affected the quality and economic benefits of fruits. Fruit tree diseases mainly include fruit diseases, stem diseases, root diseases, and leaf diseases. For example, in the case of pear tree disease caused by Potebniamyces pyri (leading to Phacidiopycnis rot of pear trees), when fruits were infected in the orchard, they typically develop the symptoms during storage. Meanwhile, this can also trigger cankers, dead bark and twig branches of pear trees (Xiao & Boal, 2005). In addition, Rosellinia necatrix caused a variety of diseases in pear trees, ranging from rotting of roots, yellowing of leaves and wilting to the eventual death of the trees (Sawant et al., 2021). As a pathogenic fungus, the genus Fusarium had the capacity to induce wilt and rot diseases, primarily affecting the roots and stem bases of a variety of fruit trees, including pears (Li et al., 2024), apples (Tang et al., 2022), and bananas (Ma et al., 2023). The rhizosphere of plants could enrich with a large number of biocontrol bacteria that can directly or indirectly inhibit the infection of plant pathogens. Therefore, clarifying the taxonomic and functions of the rhizosphere microorganisms of pear trees, and how they vary from those of the bulk soil microbiome plays an important role in exploring rhizosphere biocontrol resources. Herein, the methods of the 16S rRNA gene sequencing, integrated taxonomic data and functional analysis were used to analyze the microbial communities in the pear rhizosphere and bulk soils. Furthermore, the distribution ratio of antagonistic strains in the rhizosphere and bulk soils was evaluated with F. oxysporum as the control targets.

Materials & Methods

Study area and site selection

This study area was located in Xi County, Linfen City, Shanxi Province, northwest China (110°44′–111°15′E, 36°27′–36°85′N) (Fig. 1). This region has a planting history of ‘Yuluxiang’ pear trees. In 1999, it was named “Hometown of Chinese Golden Pears” by the Ministry of Agriculture of China. The elevation of this area ranges from 762.5 m to 1,300 m. This area is a temperate continental monsoon climate, with the annual average temperature of 9.6 °C and the average temperature in July is 23.0 °C. The annual average rainfall is 474.2 mm. The main soil types are purple-red clay and brownish-yellow sandy soil (Table 1).

Table 1:

Characteristics of the soil site.

Characteristics	Soil sample
Elevation	1,100 m
Longitude	110°96′E
Latitude	36°71′N
Slope	15°
Soil type	Purple-red clay and brownish-yellow sandy soil

DOI: 10.7717/peerj.20627/table-1

Results

Beta diversity-based comparison in pear rhizosphere and bulk soils

PCoA, NMDS and ANOSIM were used to analyze the differences in the bacterial community structure between the pear rhizosphere soil and bulk soil. The first principal coordinate (PC1, 29.13%) suggested that the bacterial communities in the rhizosphere soil differed significantly from those in the bulk soil. Additionally, PC2 also showed 10.42% inter-sample variance (Fig. 2). The NMDS result showed that the stress value was 0.062 (Fig. S2). Furthermore, the ANOSIM analysis based on effective OTUs reconfirmed that the bacterial compositions of the pear rhizosphere and bulk soils were significantly different, with an R-value of 0.731 and a p-value of 0.002 (Fig. S3).

Taxonomy-based analysis of pear rhizosphere and bulk soils

A total of 45 phyla, 544 families and 1035 genera were found in two treatments. The relative abundance of the two groups in the taxon-based evaluations was showed in Tables S4–S6. Twelve phyla contributed to the differences in the rhizosphere soil and the relative abundance of six phyla was > 1%, among which Proteobacteria was the dominant phylum and contributed over 60% of all found phyla (Figs. 3A–3B and Table S4). Compared with the rhizosphere soil, the relative abundance of Proteobacteria (from 61.24% to 46.30%), Bacteroidota (from 4.68% to 3.29%) and Firmicutes (from 4.07% to 0.90%) decreased, whereas those of Acidobacteriota (from 8.34% to 13.83%), Actinobacteriota (from 5.72% to 14.21%) and Chloroflexi (from 1.33% to 3.46%) increased in the bulk soil (Fig. 3A and Table S4). In total, 97 families and 130 genera contributed to the differences in the bacterial communities of the rhizosphere and bulk soils (Tables S5 and S6). The mean relative abundances of 12 families and 12 genera exceeded 1%. The increase in Pseudomonadaceae (from 2.12% to 3.96%) within Proteobacteria and Bacillaceae (from 0.20% to 2.19%) within Firmicutes in the rhizosphere soil was driven by a set of genera, predominantly Pseudomonas (from 2.12% to 3.94%) and Bacillus (from 0.20% to 2.16%) (Figs. 3C–3F). Thus, pear rhizosphere soil enriched the Pseudomonadaceae and Bacillaceae.

Differences in pear rhizosphere soil and bulk soil microbial phenotypes

BugBase was used to predict the coverage of functional pathways at the biological level within intricate microbiomes, and to provide biologically interpretations of the phenotypes. Herein, this method was used to analyze the phenotypic differences between the pear rhizosphere soil and bulk soil samples based on the 16S rRNA sequence results. The relative abundance of biofilm-forming microorganisms was higher in the pear rhizosphere soil sample (Fig. 4 and Table S7), mainly due to the greater abundance of Comamonadaceae, Methylophilaceae, Pseudomonadaceae, Rhodospirillaceae and Xanthomonadaceae (Fig. S4A). The relative abundance of bacteria with stress-tolerant phenotypes of the pear rhizosphere soil was significantly higher than that in the bulk soil (Fig. 4 and Table S7), which was mainly attributed to the greater abundance of Comamonadaceae, Methylophilaceae, Pseudomonadaceae and Xanthomonadaceae (Fig. S4B). The relative abundance of potential pathogenic bacteria and those with gene functions related to mobile elements were significantly more abundant in the pear rhizosphere soil sample than in the bulk soil (Fig. 4 and Table S7), which were primarily attributed to the greater abundance of taxa belonging to Bacillaceae, Comamonadaceae, Cytophagaceae, Methylophilaceae, Pseudomonadaceae, Rhodospirillaceae and Xanthomonadaceae (Figs. S4C and S4D). In addition, gram-positive, aerobic and anaerobic bacteria were less abundant in the pear rhizosphere soil than in the bulk soil (Fig. 4 and Table S7).

Analysis of antifungal capabilities of bacteria

To demonstrate the capability of plants to recruit beneficial bacteria, this experiment utilized LB medium to randomly isolate 96 cultivable clones from two groups and employed plate confrontation experiments to analyze their antifungal abilities against F. oxysporum. The results showed that a total of 41 colonies in rhizosphere soil exhibited antagonistic effects to varying degrees, accounting for 42.7%. In the bulk soil, 14 colonies showed antagonistic effects, accounting for 14.6%, which was significantly lower than the proportion of antagonistic strains in the rhizosphere soil (Tables S9 and S10). To further verify whether the antibacterial effects of these antagonistic colonies were broad-spectrum, colony of E8 from the rhizosphere soil, which exhibited significant antibacterial activity, was selected to analyze its antifungal abilities against six plant diseases, including F. acuminatum, B. cinerea, F. dermatis, S. sclerotiorum, E. turcicum and F. graminearum. We found that it had antagonistic effects against five of the tested pathogenic fungi but not against B. cinere (Fig. 7). These results demonstrated that the rhizosphere of pear trees can recruit biocontrol strains that are beneficial to plants.

Table 3:

Topological properties of networks.

Network properties	Rhizosphere soil	Bulk soil
Total nodes	655	545
Total links	615	718
Average path distance	3.519	1.000
Average degree	1.878	2.635
Average clustering coefficient	0.170	0.567

DOI: 10.7717/peerj.20627/table-3

Discussion

The rhizosphere is the soil environment where plant roots conduct their normal vital activities, and where huge amounts of microorganisms colonize. It plays an important role in maintaining plant growth and health. “Rhizosphere microorganisms” is a collective term for microorganisms that inhabit the rhizosphere environment and are influenced by root exudates (Berendsen, Pieterse & Bakker, 2012). Their abundance typically reaches 10⁹ to 10¹² microorganisms per gram of soil, which is five to 10 times higher than the number of microorganisms found in non-rhizosphere soil (Miransari, 2011; Groleau-Renaud et al., 2000). In the process of gradual evolution, recent studies have found a decreasing and selective trend in the bacterial richness and diversity of rhizosphere microorganisms in real agricultural systems (Ling, Wang & Kuzyakov, 2022). Wei et al. (2023) found that bacterial alpha diversity indices were lower in the rhizosphere soil of Chinese cabbage than that in the bulk soil. These results showed a similar trend to those findings in our study, confirming that pear rhizosphere soil and bulk soil had comparable bacterial richness and that the bulk soil had greater species diversity (Table 2, Table S3). Co-occurrence network diagrams can be utilized to study the interaction relationships among a large number of microbial species (Rottjers & Faust, 2018). Researches had revealed that in short-term farming systems, the network relationships in the rhizosphere soil of Chinese cabbage and soybean plants were simpler compared to those in the bulk soil (Wei et al., 2023; Mendes et al., 2014). In this study, the co-occurrence network in the rhizosphere soil of pear displayed a weaker structure than that in the bulk soil. The co-occurrence networks of rhizosphere soil encompassed both positive interactions (422) and negative interactions (193), whereas the bulk soil exhibited almost exclusively positive interactions. Consequently, the interactions among bacteria in bulk soil primarily were mutualistic and synergistic relationships, while the rhizosphere soil contained not only symbiotic bacterial communities but also certain bacterial communities with antagonistic, competitive and parasitic relationships. Furthermore, both the co-occurrence networks of rhizosphere soil and bulk soil contained multiple modular interaction units. In the bulk soil, these interactions involved multiple phyla, whereas in the less modular rhizosphere soil, the interactions were mostly centered around Proteobacteria (Fig. 6, Table 3). These results once again demonstrated that the bacterial community diversity in the rhizosphere soil of pear trees was lower than that in the bulk soil, and that Proteobacteria was the dominant phylum.

The diversity of bacterial communities in the rhizosphere soil was lower than that of the bacterial communities in the bulk soil. However, their compositions differed between the two soil types. The analysis results of PCoA, NMDS and ANOSIM showed that their microbial community structure and composition differed between two types of soils (Fig. 2, Figs. S2 and S3). The rhizosphere soil was characterized by Proteobacteria (61.2%), Acidobacteriota (8.3%), Actinobacteriota (5.7%), Bacteroidota (4.7%) and Firmicutes (4.1%). In contrast, Proteobacteria (46.3%), Actinobacteriota (14.2%), Acidobacteriota (13.8%), Chloroflexi (3.5%) and Bacteroidota (3.3%) were the dominant phyla in the bulk soil. Among these, compared to the bulk soil, the relative abundance of Proteobacteria and Bacteroidota were higher in the rhizosphere soil (Fig. 3A and Table S4). Ling, Wang & Kuzyakov (2022) found that Proteobacteria and Bacteroidota were capable of rapid growth in carbon-rich soil environments, and had higher metabolic activity and reproductive capacity, and were more abundant in plant rhizosphere soil than in bulk soil. Therefore, these results indicated that the rhizosphere soil of pear trees had a higher metabolic activity and nutrient availability than the bulk soil. This spontaneous environmental adaptation behavior of plants not only promoted their own growth but also had the potential to benefit soil health.

Factors influencing the structure and composition of microbial communities in the plant rhizosphere were relatively complex. The physicochemical properties of soil were the primary determinants, followed by the environmental conditions, host genotypes, and soil nutrient status (Stopnisek & Shade, 2021; Lundberg et al., 2012; Yeoh et al., 2017). Moreover, plants often developed their defense mechanisms against different pathogens by selective stimulation and support from antagonistic bacteria. Bacillus strains, including B. subtilis and B. cereus etc., represented the most promising biological control agents currently available. They primarily prevented plant diseases by producing antibacterial substances and inducing systemic resistance in plants (Jiang et al., 2016; Vlamakis et al., 2013). B. thuringiensis, a well-known insect pathogen, was also gradually being recognized for its potential in the biological control of plant diseases (Wang et al., 2020). Different Pseudomonas species could also act as beneficial to plants and insect-pathogenic roles (Vesga et al., 2020). Shen et al. (2015) found that the Bacillus and Pseudomonas genera were enriched in healthy soil of banana. In our study, the relative abundance of Bacillus and Pseudomonas were significantly increased in pear rhizosphere soil (Figs. 3C–3F). Moreover, BugBase analysis showed biofilm-forming, stress-tolerant bacteria related to disease resistance were significant increased in the pear rhizosphere soil sample (Fig. 4). However, the potential pathogenic microorganisms were also enriched in the rhizosphere (Fig. 4), which also found in other researches (Xu et al., 2021; Wei et al., 2023). The family Xanthomonadaceae, belonging to the phylum Proteobacteria, was predominantly composed of plant pathogens, including multiple pathogenic species labeled as Xanthomonas, Xylella and Stenotrophomonas. These bacteria primarily adhered to the surface of host tissues through the production of exopolysaccharides, lipopolysaccharides, and proteineous structures, thereby causing various plant diseases such as citrus canker (Mhedbi-Hajri, Jacques & Koebnik, 2011; Shahbaz et al., 2022). Notably, a minority of strains could also function as plant probiotics (Rat et al., 2021; Stork, Kim & Mudgett, 2015). The increase in the abundance of biofilm-forming, stress-tolerant and potential pathogenic phenotype-related bacteria in the pear rhizosphere soil was all associated with the rise in the abundance of Xanthomonadaceae (Fig. S4). Therefore, plants must balance their rhizosphere microbial ecosystem by different defense mechanisms.

The extensive use of chemical bactericides and other pesticides had led to issues such as pesticide residues and resistance. Therefore, there is an urgent need to explore new microbial strain resources with strong disease resistance and broad antibacterial spectrums. In our study, the analysis results of the plate confrontation experiment between bacteria collected from different ecological niches and F. oxysporum revealed that the rhizosphere soil harbored numerous bacteria with antagonistic effects (Fig. 7, Tables S9 and S10). According to literature reports, over 80% of the clones directly isolated contain multiple OTUs (Xu et al., 2024). Therefore, we further isolated bacteria from the E8 colony and conducted 16S rDNA full-length sequence analysis. The analysis results showed that the presence of strains belonging to the Bacillus and Pantoea, and further confirmed that these strains had well antagonistic effects against F. dermatis, S. sclerotiorum and F. graminearum. Notably, it was reconfirmed that Bacillus was enriched in the rhizosphere soil. In summary, these results indicated that the plant rhizosphere could recruit specific biocontrol strains, such as Bacillus. Previous research by our team had found that after insect feeding, the rhizosphere of plants enriched some strains possessing insecticidal activity (Xu et al., 2024). Herein, a high abundance of Bacillus was found in the rhizosphere soil of pear trees (Fig. 3F). Notably, B. thuringiensis, belonging to the Bacillus genus, was both an insect pathogen and capable of enhancing plant disease resistance (Jouzani, Valijanian & Sharafi, 2017; Wang et al., 2023). Therefore, further analysis should be conducted to investigate the potential of bacterial genera enriched in the rhizosphere of pear trees for controlling pear pests, such as Grapholitha molesta. It is expected to isolate the biocontrol strain resources from the rhizosphere of pear trees with the potential for pest and disease management.

Conclusions

Taken together, our results indicated that the diversity of bacteria communities in the bulk soil was higher than that in the rhizosphere soil, but their compositions differed between two types of soil. The pear rhizosphere soil exhibited a significantly higher relative abundance of bacterial phyla including Proteobacteria, Bacteroidota and Firmicutes, while Acidobacteriota, Actinobacteriota, and Chloroflexi were more prevalent in the bulk soil. The genera of rhizosphere bacteria, specifically Methylophilus, Diplorickettsiaceae, Pseudomonas, Neisseriaceae, Microscillaceae and Bacillus, were significantly higher in the pear rhizosphere. Among these genera, Bacillus and Pseudomonas were typical biocontrol strains. The relative abundance of bacteria with biofilm-forming, stress-tolerant and potential pathogenic phenotypes was significantly higher than that in the bulk soil. The FAPROTAX analysis showed the ecological functions of the rhizosphere soil were mainly affected by chemoheterotrophy, nitrogen_fixation and nitrogen_respiration. Moreover, the rhizosphere soil harbored a higher proportion of antagonistic strains. The differences in the bacterial community between the rhizosphere soil and the bulk soil provided an efficient way to exploring beneficial bacterial resources.

Supplemental Information