*Corresponding author. Phone) +86-087165228404, FAX) +86-087165228404, E-mail) Potato ranks fourth in terms of production among food crops worldwide and is widely cultivated globally (Faist et al., 2023). Yunnan is one of China’s main potato-producing regions, ranking third in both planting area and total production nationwide. However, potato plants are susceptible to infection by bacterial and fungal pathogens both before and after harvest, leading to potato diseases such as late blight, bacterial wilt, and scab, greatly limiting global consumption and development (Anwar et al., 2015; Ehiobu et al., 2022). Among these diseases, potato bacterial wilt is a serious soil-borne disease caused by Ralstonia solanacearum, with a damage level second only to that of potato late blight. It mainly occurs in tropical, subtropical, and temperate regions and is prevalent in various southern regions of China (Coll and Valls, 2013; Sebastià et al., 2021). Up to 100% bacterial wilt incidence rates have been observed in field parcels of some highly affected areas, causing an annual yield loss of 10-15%. Severely diseased fields can experience yield reductions of up to 80% or even complete crop failure, posing a serious threat to the healthy development of the potato industry in certain region (Li et al., 2021, 2024).
R. solanacearum is considered the second most common bacterial plant pathogen in the world and can cause devastating bacterial wilt in more than 200 plant species across 54 families (Coll and Valls, 2013; Mansfield et al., 2012). Importantly, R. solanacearum can survive in soil for an extended period (up to 40 years) even without a host plant. Under suitable conditions, R. solanacearum can infect plants through their roots, colonize xylem tissues, and thus block water flow and cause plant wilting, making disease management almost unattainable (Ahmed et al., 2022c; Genin and Denny, 2012). The rhizosphere is a critical microdomain for plant-soil-microbe interactions and is closely associated with the colonization and successful infection of plants by soil-borne pathogens (Zheng et al., 2022). Rhizosphere microbial communities are considered the first line of defense against soil-borne pathogens and an important determinant of plant health (Li et al., 2021; Wen et al., 2022; Yang et al., 2023). Recent studies have demonstrated that plants recruit specialized microbiomes by releasing volatile organic compounds or altering the synthesis and secretion of specific root exudates to antagonize pathogens or modulate the host immune system as adaptive strategies for growth promotion and disease suppression (Schulz-Bohm et al., 2018; Trivedi et al., 2020; Yuan et al., 2018). Moreover, research has revealed the dynamic changes in and assembly of plant-associated microbial communities in response to pathogen stress, with the host health largely affected by complex dynamic interactions among the host, microbes, and the environment (Sessitsch et al., 2019; Yang et al., 2024). However, little is currently known about the assembly and functional adaptive changes in potato rhizosphere microbial communities at the different stages of R. solanacearum infection.
Currently, advancements in science and modern sequencing tools have made the study of host-microbe interaction easier (Ahmed et al., 2022a). Amplicon sequencing can provide high taxonomic resolution and is commonly used for the analysis of the taxonomic composition of communities in samples. Metagenomic sequencing provides a holistic view of the microbial community by sequencing all the genetic material in a sample. This includes not only taxonomic information but also functional genes and pathways, offering insights into the metabolic capabilities and interactions within the community. Therefore, the combination of amplicon and metagenomic sequencing is beneficial for a more comprehensive understanding of microbial community dynamics, while also further revealing the functional potential of microbial groups, including their potential interactions with the environment and other organisms (Arevalo et al., 2019; Berg et al., 2020).
In this study, we hypothesized that R. solanacearum stress leads to reassembly and functional adaptive changes in potato rhizosphere microbial communities and that the composition and ecological functions of potato rhizosphere microbial communities vary at different stages of R. solanacearum infection. To test these hypotheses, we used amplicon (bacteria and fungi) and metagenomic sequencing technologies to explore the differences in the composition and functions of potato rhizosphere microbial communities at the four stages of R. solanacearum infection. Our findings will help better elucidate the reassembly strategies of host plant rhizosphere microbial communities under pathogen stress, which is of significant importance for harnessing rhizosphere microbial communities to enhance plant health and maximize crop yield.
Materials and Methods
Microorganisms and plant resources
The pathogen R. solanacearum used in this study was provided by the Institute of Agricultural Environment Resources, Yunnan Academy of Agricultural Sciences. After the R. solanacearum strain was activated, it was inoculated in Luria-Bertani broth, grown overnight with shaking, and then diluted with sterile distilled water to a cell concentration of 1 × 108 cfu/ml.
The potato variety used in this study was “Hezuo 88”. Healthy potato cubes were selected and planted in plastic pots filled with disease-free red soil to a depth of about 15-20 cm. All potted plants were placed in plastic greenhouses at the Institute of Agricultural Environment Resources, Yunnan Academy of Agricultural Sciences, with a day/night air temperature of 26°C/15°C and 30-65% humidity. The plants were sprayed with water 3-4 times per week and were not fertilized. After the potato plants had grown, vigorous potato plants with consistent growth were selected as test materials.
Pathogen inoculation and soil sampling
To ensure the incidence and avoid the disturbance of soil microorganisms caused by the root irrigation treatment with the pathogenic bacteria, we chose to use the capillary injection method to inoculate 1 ml of R. solanacearum suspension into the potato stems. The specific inoculation method was as follows: Healthy one-month-old potato plants were selected, a 0.5-mm capillary tube was used to draw 1 ml of R. solanacearum suspension and inserted into the young, tender part of the potato stem at a 45° angle. The capillary tube was slowly withdrawn after all of the bacterial suspension in the capillary tube had been injected into the potato stem. In addition, to exclude the influence of other pathogenic factors in the soil or environment, we simultaneously inoculated an equal amount of sterile water as a control. The inoculated potato plants were transferred to a plastic greenhouse for cultivation, and the disease status of the potato plants was observed every day. Based on the symptoms after R. solanacearum infection (Fig. 1), we collected potato rhizosphere soil samples at four selected key time points representing different infection stages. These time points were R. solanacearum infection for 1 day (T1QK), 3 days (T3QK), 7 days (T7QK), and 15 days (T15QK). A total of four treatments were included, with three replicates per treatment and each replicate consisting of three inoculated plants.
At each sampling stage, the tested potato plants were uprooted, and after the loose soil was shaken off, the soil adhering to the roots was carefully collected using a brush and considered the rhizosphere soil. The rhizosphere soil from the three inoculated plants in each replicate was pooled as a mixed sample, resulting in a total of 12 samples. The mixed samples were placed in 10 ml sterile conical tubes, snap-frozen in liquid nitrogen, and immediately stored in a −80°C freezer for later use.
Amplicon analysis
Microbial DNA extraction, PCR amplification, and sequencing
The DNA from 12 rhizosphere soil samples was extracted following the steps of the FastDNA SPIN Kit for Soil (MP Biomedical, Carlsbad, CA, USA) soil genomic DNA extraction kit. The DNA concentration and purity were measured using a NanoDrop 2000, and the quality of the extracted DNA was examined by 1% agarose gel electrophoresis. Using the DNA extracted above as a template, two pairs of universal primers, 338F (5′-ACTCCTACGGGAGGCAGCAG-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′), as well as ITS1 F (5′-ACTTGGTCATTTAGAGGAAGTAA-3′) and ITS2 R (5′-BGCTGCGTCTTCATCGATGC-3′), were used to amplify the V3-V4 variable region of the bacterial 16S rRNA gene and the ITS1 region of fungal rRNA gene, respectively. The recovered products were purified using an AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, USA), measured by 2% agarose gel electrophoresis, and tested and quantified using a Quantus Fluorometer (Promega, Madison, WI, USA). Amplicon libraries were sequenced on the Illumina HiSeq 2500 platform in Shanghai Majorbio Bio-Pharm Technology Co., Ltd. (Shanghai, China).
Quality control and analysis of the microbiome data
Quality control of paired-end raw sequencing reads was performed using Fastp (version 0.19.6) (Chen et al., 2018), and paired-end sequences were assembled using FLASH software (version 1.2.11) (Magoč and Salzberg, 2011). The quality-controlled and assembled sequences were then clustered into operational taxonomic units (OTUs) at a 97% similarity threshold using UPARSE software (version 11), and chimeras were excluded (Edgar, 2013). The taxonomic annotation of OTUs was conducted using the RDP classifier (version 2.13), aligning against the SILVA 16S rRNA gene database (version 138) (Quast et al., 2013) and UNITE Fungal ITS database (version 8.0) (Kõljalg et al., 2005) with a confidence threshold of 70%. Alpha diversity indices, including Chao 1 and Shannon, were calculated using Mothur software (version 1.30.2), and the Wilcoxon rank sum test was used to analyze the differences in alpha diversity among groups. Principal coordinate analysis (PCoA) based on the Bray-Curtis distance algorithm was performed to examine the overall changes in microbial community structure among the samples. Additionally, the permutational analysis of variance (PERMANOVA) nonparametric test was employed to determine whether there was a significant difference in the microbial community structure between the sample groups. Utilize Circos-0.67-7 software to construct a relationship diagram between Circos samples and species, to display the distribution of microbial species present in different microbial samples.
Metagenomic analysis
DNA extraction and sequencing
Total genomic DNA was extracted from 12 samples using the E.Z.N.A. Soil DNA Kit (Omega Bio-tek, Norcross, GA, USA) according to manufacturer’s instructions. DNA extract quality was checked on 1% agarose gel. Then, DNA extract was fragmented to an average size of about 400 bp using Covaris M220 (Gene Company Limited, China) for paired-end library construction. Paired-end library was constructed using NEXTFLEX Rapid DNA-Seq (Bioo Scientific, Austin, TX, USA). Adapters containing the full complement of sequencing primer hybridization sites were ligated to the blunt-end of fragments. Paired-end sequencing was performed on Illumina Novaseq 6000 (Illumina Inc., San Diego, CA, USA) at Majorbio Bio-Pharm Technology Co., Ltd.
Bioinformatics analysis of metagenomic sequencing data
The adapter sequences at the 3′ and 5′ ends of the reads were quality-trimmed using fastp to remove reads with a length less than 50 bp, an average base quality below 20, or the presence of N bases. The filtered sequences were assembled using MEGAHIT (version 1.1.2) (Li et al., 2015), and contigs with a length ≥ 300 bp were selected as the final assembly results. Open reading frames were predicted for the assembled contigs using Prodigal (version 2.6.3) (Hyatt et al., 2010). The genes with nucleic acid lengths greater than or equal to 100 bp were selected, and the gene sequences predicted from all samples were clustered using CD-HIT software (version 4.6.1) (Fu et al., 2012). The longest gene in each class was taken as the representative sequence to construct a non-redundant gene set. The amino acid sequences of the non-redundant gene set were compared with those in the NR database (version nr_202209) using Diamond (version 2.0.13), and the species annotations were obtained from the taxonomic information database corresponding to the NR database. Then, the abundance of each species was calculated using the sum of the abundances of genes corresponding to the species (Buchfink et al., 2015). The functional profiles including Kyoto Encyclopedia of Genes and Genomes (KEGG) Orthology (KO), cluster of orthologous groups of proteins (COG), and carbohydrate-active enzymes (CAZy) annotation was conducted using Diamond against the Kyoto Encyclopedia of Genes and Genomes database (version 202109), COG database (version 2020) and CAZy database (version 8). To estimate the relative abundance of metagenome-assembled genomes (MAGs), the short-read sequences were mapped to the non-redundant gene catalog with 95% identity using SOAPaligner (version 2.21) (Li et al., 2008), and read counts were normalized to per kilobase per million mapped reads (RPKM) using CoverM (version 0.4.0).
Results
Evaluation of the microbial community structure and community diversity in the potato rhizosphere during different infection stages
Through high-throughput sequencing, 829,862 effective bacterial sequences and 997,131 effective fungal sequences were obtained from 12 rhizosphere soil samples, with average lengths of 417 bp and 240 bp, respectively. Classification annotation of the effective reads was performed at the 97% clustering level, resulting in a total of 8,741 bacterial OTUs and 2,170 fungal OTUs identified from the 12 samples. The Venn diagrams in Fig. 2A and B show the numbers of shared and unique OTUs in the different treatment groups. There were 2,665 bacterial OTUs and 458 fungal OTUs shared among the treatments (Fig. 2A and B), accounting for 30.49% and 21.11% of the total OTUs, respectively. The numbers of bacterial and fungal OTUs unique to the T3QK, T7QK, and T15QK treatments were greater than those in the T1QK treatment. The number of unique bacterial and fungal OTUs in the potato rhizosphere soil was the highest after 15 days of infection with the bacterial wilt pathogen (T15QK) and was 26.75%, 14.45%, and 14.71% (bacteria) and 53.55%, 46.61%, and 11.34% (fungi) higher than those in T7QK, T3QK, and T1QK, respectively.
Based on PCoA using the Bray-Curtis distance algorithm, we evaluated the impact of bacterial wilt pathogen on the microbial community structure of potato rhizosphere soil (Fig. 2C and D). The percentages of variance explained by principal coordinate 1 (PC1) and principal coordinate 2 (PC2) were 33.21% and 16.96% (bacteria) and 56.41% and 17.17% (fungi), respectively, totaling 50.17% and 73.58% of the samples, respectively. PERMANOVA analysis showed that the impact of R. solanacearum on the potato rhizosphere fungal communities was greater than that on the rhizosphere bacterial communities (R2 = 0.35 for bacteria and R2 = 0.861 for fungi, P ≤ 0.001 for both). When comparing different treatments, we found that the duration of R. solanacearum infection were separated in terms of the second principal component. The T1QK and T3QK samples (bacteria and fungi) were distributed mainly on the upper side of the PC2 axis, while T7QK and T15QK samples (bacteria and fungi) were distributed mainly on the lower side of the PC2 axis. In addition, we observed a distinct separation of samples from T15QK compared to samples from the other three treatment groups, suggesting that R. solanacearum infection and the duration of infection may be the main factors affecting the microbial community structure in potato rhizosphere soil.
We further evaluated the changes in the diversity indices of bacterial and fungal communities in the potato rhizosphere soil after different durations of infection (α-diversity indices: Shannon and Chao 1). First, in terms of the bacterial communities, the Shannon indices for T1QK, T3QK, and T7QK gradually decreased, with the Shannon index for T15QK being significantly higher than that for T1QK, T3QK, and T7QK (P < 0.01) (Fig. 3A). The Chao1 index of the bacterial communities was higher for T3QK and T15QK than for T1QK and T7QK; the Shannon and Chao1 indices for T7QK were lower than those for the other three treatments (Fig. 3B). With respect to the fungal communities, compared with that for T1QK, the Shannon index for T3QK was significantly lower, that for T7QK was significantly higher, and that for T15QK was not significantly different (Fig. 3C). The Chao1 index gradually increased with increasing duration of infection from T1QK to T15QK (Fig. 3D).
Species composition of potato rhizosphere microbial communities at different infection stages
Next, we analyzed the species composition of potato rhizosphere microorganisms at different infection stages at different taxonomic levels. At the phylum level, Proteobacteria, Actinobacteria, Firmicutes, Chloroflexi, and Acidobacteria were the main dominant bacterial phyla in the potato rhizosphere soil at different infection stages, while Ascomycota and Mortierellomycota were the most dominant fungal phyla (Fig. 4A and B). In the bacterial communities, there was no significant difference in the relative abundance of Proteobacteria or Actinobacteria among the four treatments (P > 0.05); there was no significant difference in the relative abundance of Firmicutes in the T1QK (14.21%) and T3QK (13.06%) groups (P > 0.05), but the relative abundance was significantly higher in T7QK (20.40%) than in those two treatments (P < 0.05). As the infection time increased, the relative abundance in T15QK (6.04%) decreased significantly compared to that in the other three treatments (P < 0.05). Similarly, there was no significant difference in the relative abundance of Chloroflexi between T1QK (9.50%) and T3QK (9.36%) (P > 0.05); the relative abundance in T7QK (8.07%) was significantly lower than that in the former two treatments (P < 0.05), and the relative abundance in T15QK (13.17%) was significantly higher than that in the first three treatments (P < 0.05). In terms of the fungal communities, the relative abundance of Ascomycota in T3QK (78.74%) and T15QK (74.70%) was greater than that in T1QK (70.43%) and T7QK (68.09%). In contrast, the relative abundance of Mortierellomycota in T3QK (8.08%) and T15QK (13.35%) was lower than that in T1QK (15.44%) and T7QK (17.69%).
The Circos sample-species relationship diagrams in Fig. 4 illustrate the dominant bacterial and fungal communities at the genus level among the different treatment groups. Among the bacterial communities, Bacillus, Arthrobacter, Pseudomonas, and Sphingomonas were dominant in the different groups (Fig. 4C). Among the different treatment groups, the relative abundance of Bacillus in the T3QK (7.42%) and T15QK (3.05%) groups was significantly lower than that in the T1QK group (9.80%) (P < 0.05), while the relative abundance in the T7QK group (13.02%) was significantly higher than that in the T1QK group (P < 0.05). The relative abundance of Arthrobacter was the lowest in the T1QK (6.00%) group, and the relative abundance in the T3QK (9.90%), T7QK (7.60%), and T15QK (7.25%) groups was higher than that in the T1QK group; the relative abundance was highest in the T3QK group. The relative abundance of Pseudomonas in T3QK (4.28%) was higher than that in T1QK (4.00%), while that in T7QK (1.61%) and T15QK (0.73%) was lower than that in T1QK. The relative abundance of Sphingomonas increased gradually with increasing infection time, from 2.20% in T1QK to 3.12% in T15QK. Among the fungal communities, Humicola, Mortierella, and Chaetomium were the dominant communities in the different groups (Fig. 4D). The relative abundances of Humicola and Chaetomium were significantly higher in the T3QK (27.70%, 11.54%) and T15QK (21.51%, 6.96%) groups than in the T1QK group (9.12%, 6.17%) (P < 0.05). In contrast, the relative abundance of Mortierella in the T3QK (8.03%) and T15QK (13.10%) groups was lower than that in the T1QK group (15.38%). These results indicated that the bacterial and fungal communities in the potato rhizosphere changed dynamically in response to stress during the different stages of R. solanacearum infection.
Functional composition of potato rhizosphere microbial communities at different infection stages
To further elucidate the ecological functions of bacterial and fungal communities in the potato rhizosphere during the different stages of R. solanacearum infection, we performed metagenomic sequencing on 12 samples. The number of raw reads obtained varied between 89,160,956 and 111,887,670. Following a quality assessment, the clean reads ranged from 87,462,474 to 109,740,136. The reads were then assembled to an average of approximately 604,760 contigs and ranged from 226,029,053 to 537,522,321 bp (Supplementary Table 1). The metagenomic sequencing data were assigned to 33,036 bacterial species and 1,610 fungal species. Basically consistent with the amplicon sequencing data, the bacterial community primarily included species from Proteobacteria, Actinobacteria, Firmicutes, Chloroflexi, Acidobacteria, Gemmatimonadetes, and Bacteroidota, while the fungal community was mainly composed of species from Basidiomycota, Mucoromycota, and Ascomycota (Supplementary Fig. 1). Here, we observed a substantial difference in the relative abundance of Ascomycota and Basidiomycota between the data obtained from high-throughput sequencing and metagenomic sequencing. We speculate that this discrepancy arises from a combination of factors, such as the distinct specificities of the two sequencing approaches, variations in sequencing depth and coverage, and discrepancies among the species annotation databases employed.
Nonmetric multidimensional scaling (NMDS) ordination analysis showed significant differences in the composition of KO, COG, and CAZy functions in the potato rhizosphere microbial communities during different stages of R. solanacearum infection (R2 = 0.684 for KO, R2 = 0.687 for COG, R2 = 0.686 for CAZy, P < 0.01). In particular, the T15QK samples were distinctly separated from the samples of the other three treatment groups, indicating substantial changes in the microbial community functions of the T15QK treatment group (Fig. 5A, Supplementary Fig. 2A and B). The T3QK, T7QK, and T15QK treatment groups exhibited greater diversity of COG and CAZy functions than the T1QK treatment group (based on the Shannon index), while the Shannon indices of KO, COG, and CAZy functions gradually decreased as the infection time increased from T3QK to T15QK (Fig. 5B).
We performed differential abundance analysis to determine the functional characteristics of potato rhizosphere microbiomes during different infection stages. First, in terms of CAZy functions, the relative abundance of genes such as sucrose synthase (GT4), UDP-GlcNAc: peptide beta-N-acetylglucosaminyltransferase (GT41), N-acetylglucosamine 6-phosphate deacetylase (CE9), and gluco-oligosaccharide oxidase (AA7) were higher in the T3QK, T7QK, and T15QK treatment groups than in the T1QK group; the relative abundance of each of these genes was highest in the T3QK treatment group, followed by T15QK and T7QK. In contrast, the relative abundance of genes such as lysozyme G (GH23), β-glucosidase (GH1), lipopolysaccharide N-acetylglucosaminyltransferase (GT9), α-glucosidase (GH31), glycogen or starch phosphorylase (GT35), β-galactosidase (GH2), and cellobiose phosphorylase (GH94) were lower in the T3QK, T7QK, and T15QK treatment groups than in T1QK (Fig. 5C). In terms of COG functions, the relative abundances of genes in modules such as the signal transduction mechanism (COG_T), energy production and conversion (COG_C), replication, recombination and repair (COG_L), and secondary metabolite biosynthesis, transport and catabolism (COG_Q) modules were higher in the T3QK, T7QK, and T15QK treatment groups than in T1QK. As the infection time increased from T1QK to T15QK, the relative abundance of genes in the coenzyme transport and metabolism (COG_H) module gradually increased. Among the four treatment groups, the T15QK treatment group had the lowest relative abundance of genes in the cell wall/membrane/envelope biogenesis (COG_M) module and the highest relative abundance of genes in the defense mechanism (COG_V) module (Fig. 5D).
As is well known, soil microbes participate in a variety of ecosystem functions, including the decomposition of organic matter and the cycling of nutrients, playing a critical role in the transformation of soil nutrients such as C, N, and P. We further analyzed and summarized the genes involved in C metabolism, N metabolism, and the P cycling in this study. Heatmaps of the levels of related genes’ abundance during different infection stages are shown in Fig. 6. We found that as the infection time increased, the abundance of genes involved in C, N, and P metabolism in the potato rhizosphere microbial communities tended to decrease. For example, C metabolism-related genes such as 2-dehydro-3-deoxygluconokinase (kdgK), isocitrate lyase (aceA), phosphogluconate dehydratase (edd), and phosphine phosphatase (serB) and N metabolism-related genes such as nitrate reductase/nitrite oxidoreductase (narG), nitrite reductase (NADH) large subunit (nirB), nitrate/nitrite transporter (narK), nitrate reductase/nitrite oxidoreductase (narH), assimilatory nitrate reductase catalytic subunit (nasA), and NADH small subunit (nirD) all showed lower relative abundance in the T3QK, T7QK, and T15QK treatment groups than in T1QK. In particular, we noticed that the relative abundances of the narK, nirD, edd, nitrite reductase (NO-forming)/hydroxylamine reductase (nirS), phosphate transport system permease protein (pstA), and acid phosphatase (phoN) genes were the lowest in the T15QK treatment group among the four treatments.
In summary, the above results indicated that R. solanacearum infection led to substantial changes in the relevant functions of the potato rhizosphere microbial communities. The functional diversity of the potato rhizosphere microbiome gradually decreased as the infection time increased. In the treatment group with the most severe infection, the functional genes related to signal transduction mechanisms, energy production and conversion, and defense mechanisms were enriched, but the abundance of functional genes related to C, N, and P metabolism decreased significantly.
Discussion
Interactions between plants and associated microbial communities play a crucial role in promoting the productivity and health of plants in natural environments (Mendes et al., 2011; Vandenkoornhuyse et al., 2015). The health of the host is largely affected by complex dynamic interactions among the host, microbes, and environment. Elucidation of the assembly and ecological functions of host plant-associated microbial communities under pathogen stress is essential for the application of related microbial communities in the sustainable future improvement of agricultural productivity (Sessitsch et al., 2019).
In this study, we first analyzed the effects of four stages of R. solanacearum infection on the assembly of the potato rhizosphere microbiome. We found that R. solanacearum infection and the duration of infection explained the significant variations in the potato rhizosphere microbial communities. Based on PCoA using the Bray-Curtis distance algorithm, we found the separation of R. solanacearum infection duration along the second principal component. In particular, the samples taken after 15 days of R. solanacearum infection showed distinct separation from the other three treatments, implying significant differences in the structure of the potato rhizosphere microbial communities across different stages of R. solanacearum infection. In addition, we observed a gradual decrease in the Shannon index of bacterial communities from T1QK to T3QK to T7QK, but the Shannon index of T15QK was significantly higher than that of the other three treatments. With increasing duration of R. solanacearum infection, the Chao1 index of the fungal communities gradually increased from T1QK to T15QK. These findings are consistent with previous studies, indicating the dynamic changes in the bacterial and fungal communities of the potato rhizosphere in response to stress during different stages of R. solanacearum infection (Ahmed et al., 2022b; Gao et al., 2021; Yang et al., 2023).
Further analysis of the composition of the bacterial and fungal communities in the potato rhizosphere at different infection stages revealed that 2,665 and 458 core bacterial and fungal OTUs coexisted in potato rhizosphere soil samples at different infection stages. We observed significant differences in some dominant bacterial and fungal communities during different infection stages. For example, the relative abundances of Firmicutes and Chloroflexi were not significantly different between T1QK and T3QK, while T7QK showed significantly higher relative abundance of Firmicutes and significantly lower relative abundance of Chloroflexi than did the former two groups. We observed the lowest relative abundance of Firmicutes and the highest relative abundance of Chloroflexi in the T15QK treatment group. Studies have shown that Firmicutes is associated with C source utilization and can degrade cellulose, lignin, and wood fibers by secreting hydrolytic enzymes, while Chloroflexi is associated with the utilization of organic halides (Wang et al., 2020). Metagenomic sequencing revealed that functional genes involved in C metabolism were significantly reduced in potato rhizosphere microbes at the most severely infected stage (T15QK). Additionally, it has been previously reported that the specific destruction of protective Actinobacteria and Firmicutes communities in the tomato rhizosphere increases the incidence of bacterial wilt (Lee et al., 2021). At the genus level, many strains of Bacillus and Pseudomonas have been reported to produce broad-spectrum antibacterial compounds to inhibit the growth of pathogens, colonize host plants, and induce systemic disease resistance in plants; they are thus widely used as biological control agents against soil-borne diseases (Ahmed et al., 2022b). Here, we found that the relative abundance of Bacillus in the T3QK and T15QK groups was significantly lower than that in T1QK, while the relative abundance in the T7QK group was significantly higher than that in T1QK. Moreover, the relative abundance of Pseudomonas was higher in T3QK than in T1QK, while that in T7QK and T15QK were lower than that in T1QK. Certain strains of Sphingomonas are closely related to N fixation and can enhance plant survival under environmental stress by improving the soil environment and degrading toxic substances (Xie and Yokota, 2006). Interestingly, in this study, the relative abundance of Sphingomonas gradually increased as the infection time increased, with the relative abundance being the highest in the most severely infected T15QK group. In terms of fungal communities, Ascomycota contains a large number of pathogens that are not pathogenic to crops and usually aggregate in plant roots, causing damage to the root surface and creating conditions for infection by certain pathogenic bacteria (Yang et al., 2023). Here, we found that the relative abundance of Ascomycota in T3QK and T15QK was higher than that in T1QK and T7QK. Species of Mortierella have been reported to be associated with the suppression of soil-borne diseases and to participate in N transformation in the soil (Wang et al., 2022). In this study, we revealed that the relative abundance of Mortierella was lower in T3QK and T15QK than in T1QK. These findings further support that the infection of R. solanacearum can result in significant alterations in the composition of the potato rhizosphere microbial community. This may be an indirect result of changes in potato root exudates following pathogen invasion. Previous studies have also highlighted that pathogen-induced changes in root exudation profiles may serve to control pathogens both by direct inhibition and by indirectly shifting the composition of the rhizosphere microbiome (Gu et al., 2016). In the future, we will integrate additional experiments to delve deeper into the mechanisms of rhizosphere microbiota restructuring in potatoes under pathogen invasion.
In addition to community composition, the ecological functions of potato rhizosphere microbial communities were found in this study to change during different infection stages. Similar to the microbial community composition, the KO, COG, and CAZy functional compositions of the T15QK samples were distinctly separated from those of the other three treatments on the NMDS2 axis, indicating substantial changes in the microbial community functions of the T15QK treatment group. The T3QK, T7QK, and T15QK treatment groups exhibited greater COG and CAZy functional diversity than did T1QK. However, T15QK had lower KO, COG, and CAZy functional diversity than did T3QK and T7QK. We observed that as the duration of infection increased (from T3QK to T15QK), the relative abundance of genes involved in signal transduction mechanisms, energy production and conversion, replication, recombination and repair, and the biosynthesis, transport, and catabolism of secondary metabolites in the potato rhizosphere significantly increased compared to that in T1QK. Among the four treatments, the T15QK treatment group had the lowest relative abundance of genes associated with cell wall/membrane/envelope biogenesis and the highest relative abundance of genes related to defense mechanism modules. These results indicated that the duration of R. solanacearum infection significantly affected the basic functions of the potato rhizosphere soil microbial communities and the information transfer process with the surrounding environment, thereby influencing the ecological effects of microbial communities in the soil environment (Yang et al., 2023).
C, N, and P in the soil are three key nutrient elements for soil fertility and plant growth and development. Soil microbes often participate in the transformation of C, N, and P through their metabolic activities (Wu et al., 2016; Xiong et al., 2021). A recent study has shown that the rice rhizosphere bacterial microbiota can profoundly affect the N use efficiency of host plants (Zhang et al., 2019). Here, we found that compared with that in T1QK, the relative abundance of N cycle-related enzyme genes (such as β-glucosidase and α-glucosidase) and N metabolism-related genes (such as nitrate reductase/nitrite oxidoreductase, NADH large subunit, and nitrate reductase/nitrite oxidoreductase) in the rhizosphere microbial communities in the T3QK, T7QK, and T15QK treatment groups decreased with increasing duration of infection. In particular, in the most severely infected T15QK treatment group, the relative abundance of C, N, and P cycle-related genes, such as phosphogluconate dehydratase, nitrate/nitrite transporter, NADH small subunit, and nitrite reductase (NO-forming)/hydroxylamine reductase, was the lowest among the four treatments. Previous studies have shown that the occurrence of soil-borne diseases is closely related to the imbalance of the plant rhizosphere microecology. On one hand, the imbalance of soil nutrients (such as C, N, and P) can lead to nutrient deficiencies or toxicity, weakening plant defenses and making plants more susceptible to diseases (Huang et al., 2020; Liu et al., 2016). On the other hand, the metabolism of C, N, and P by soil microorganisms also affects the composition and activity of the soil microbial community. Changes in the metabolism of carbon, nitrogen, and phosphorus in the soil can lead to nutrient competition among different microbial groups, affecting the balance between beneficial and pathogenic microorganisms, and thus leading to disease outbreaks (Cai et al., 2021; Wang et al., 2020). Overall, as the duration of potato infection by R. solanacearum increased, the rhizosphere microbial communities played a significant role in basic metabolism and signal transduction, but the functions involved in soil C, N and P transformation weakened, suggesting an adverse shift in the potato rhizosphere microecological environment.
Through the investigation of the temporal dynamics of the potato rhizosphere bacterial and fungal communities during the four stages of R. solanacearum infection, we have gained a deeper understanding of the assembly and functional adaptability of the potato rhizosphere microbiome under pathogen stress. Our results showed that R. solanacearum infection led to significant shifts in the composition and functions of the bacterial and fungal communities in the potato rhizosphere. The relative abundance of some beneficial microorganisms in the potato rhizosphere, including Firmicutes, Bacillus, Pseudomonas, and Mortierella, decreased as the duration of infection increased. Moreover, the related microbial communities played a significant role in basic metabolism and signal transduction; however, the functions involved in soil C, N, and P transformation weakened. These results further support the dynamic changes in the composition and functions of potato rhizosphere microbial communities across different stages of R. solanacearum infection to adapt to the growth promotion or disease suppression strategies of the host plant. These findings further deepen our understanding of the reassembly strategies of host plant rhizosphere microbial communities under pathogen stress and contribute toward efforts to explore and utilize functional microbial resources.
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