Diversity and Recruitment Strategies of Rhizosphere Microbial Communities by Camellia fascicularis, a Plant Species with Extremely Small Populations in China: Plant Recruits Special Microorganisms to Get Benefit out of Them

Abstract

Camellia fascicularis belongs to the family Theaceae and is a plant species with extremely small populations. It is also a second-class national protected plant in China. In recent years, the anti-inflammation, antioxidation, and antitumor effects of C. fascicularis polyphenols and flavonoids have been reported. However, changes in the soil chemistry and microbes after artificial cultivation of C. fascicularis have not been well studied. Therefore, three healthy plants from each different artificial planting year’s plot (Age_3, Age_5, and Age_7) were selected, and the chemical properties of the rhizosphere soil and root endophytic microbial communities for different cultivation years of C. fascicularis were studied in Hekou County, China. The accumulation of pathogenic and beneficial microbes in the rhizosphere of C. fascicularis was also discussed. The results show that (1) the alpha diversity in rhizosphere soil was significantly higher than that in roots, and roots recruited more Actinobacteria, which might produce beneficial secondary metabolites for the plant; (2) the total nitrogen in the rhizosphere soil of C. fascicularis cultivated for 7 years was significantly higher than that in the soil cultivated for 3 years; (3) there was no significant difference in the alpha and beta diversity in the rhizosphere soil and root endophytes of C. fascicularis in different cultivation years; (4) there was no difference in the abundance of plant-growth-promoting rhizobacteria (PGPR) in either the rhizosphere soil or roots, but the number of PGPR in roots was higher than that in rhizosphere soil; and (5) the changes in pathogenic fungi and biocontrol fungi in rhizosphere soil were greater than those of endophytic fungi in roots. The results show that there are no significant differences in microbial communities among 3, 5, and 7 years, but the influence of the outside environment on the soil and fungi was greater than that of the roots and bacteria. These results can help us to understand the soil chemical and microbial community changes during the artificial cultivation of C. fascicularis and play an important role in its artificial conservation and breeding, as it is a plant species with extremely small populations.

1. Introduction

Camellia fascicularis Hung T. Chang (Theaceae) is distributed in Gejiu City, Maguan County, and Hekou County of Yunnan Province, and approximately 663 wild single plants have been listed as second-class national protected plants in China [1]. According to the IUCN evaluation criteria, C. fascicularis belongs to the ‘critically endangered’ grade [1]. In recent years, anti-inflammation, antioxidation, and antitumor effects of C. fascicularis polyphenols and flavonoids have been reported [1,2,3]. Therefore, the artificial cultivation of C. fascicularis is not only beneficial to its restoration and protection as a plant species with extremely small populations (PSESPs), but also provides a material guarantee for the development and utilization of C. fascicularis resources in the future.

The cultivation of C. fascicularis has been carried out in Hekou County, Yunnan Province, for over 7 years. However, the soil conditions of C. fascicularis after artificial cultivation have not been well studied. After artificial cultivation, C. fascicularis inevitably accumulates pathogens in its growing areas, which may have adverse effects on plant hosts [4]. At the same time, it may also accumulate beneficial microbes and play ecological roles in promoting growth, nutrient uptake, stress tolerance, and resistance to pathogens [5]. Therefore, understanding the changes in the soil chemical properties and microbial communities in the rhizosphere of C. fascicularis has a positive effect on understanding the adaptability of C. fascicularis to artificial cultivation.

It has been reported that the bacterial and fungal communities in the roots of plants are not completely random or simply based on the distribution of the abundance gradient of rhizosphere soil; they are the result of a screening and enrichment process mediated by root exudates [6]. Although there are many bacterial groups in nature, the distribution and abundance of the main bacterial groups in the soil are similar. The main bacteria belong to the phyla Proteobacteria, Acidobacteria, Actinobacteria, and Bacteroidetes [5]. However, the number of endophytic bacteria in plant roots is very different, with Proteobacteria and Firmicutes being more than twice as abundant in roots as compared to rhizosphere soil, whereas Bacteroidetes are less abundant relative to rhizosphere soil [5]. Rhizosphere soil and root fungi comprise mainly Ascomycetes and Basidiomycetes, but the fungal communities in soil and plant roots seem to be more affected by random changes and respond differently to environmental factors than bacteria [7].

Functionally, some studies have demonstrated that the bacterial microbiota is essential for plant survival and for the protection of root-derived filamentous eukaryotes, revealing that rhizosphere bacteria positively affect plant hosts; the effect of fungi is negative [8]. Rhizosphere-accumulated plant root exudates, such as amino acids and sugars, provide a rich source of energy and nutrients for bacteria in this narrow area of soil affected by the root system; the number of bacteria is usually 10 to 100 times higher than in bulk soil [9,10]. Therefore, plant-growth-promoting rhizobacteria (PGPR) are able to provide cross protection against multiple stress factors and facilitate growth of their plant symbionts in many ways, including biofertilization, stimulation of root growth, and rhizoremediation [11,12]. Enterobacter, Pseudomonas, Bacillus, Streptomyces, Paenibacillus, and Burkholderia are the PGPR genera in C. sinensis that perform functions such as phosphate solubilization, IAA production, siderophore production, ammonia production, immunomodulatory activity, nitrogen fixation, and 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity [13]. Arbuscular mycorrhizal fungi (AMF) belong to the phylum Glomeromycota, and through symbiosis with more than 80% of higher plant roots in terrestrial ecosystems, form symbionts that can increase the uptake of nutrients including nitrogen (N), phosphorus (P), and silicon (Si), as well as increase host resistance to various stresses [14]. In addition, fungi are one of the main sources of pathogens. Colletotrichum [15,16,17], Pestalotiopsis [16,17], Exobasidium [18], Botryodiplodia [16], Macrophoma [19], Fusarium [20], Corticium [20], Ustulina [20], and Sphaerotheca [20] are major pathogenic fungi for C. sinensis. Plant growth is a combination of these effects and involves a dynamic equilibrium for both beneficial and harmful microbes.

In this study, we collected the rhizosphere soil and roots of C. fascicularis from different years of cultivation in Hekou County, China. The microbial communities of the rhizosphere soil and endophytic communities of C. fascicularis roots were detected using high-throughput sequencing. Then, the changes in PGPR bacteria, pathogenic fungi, and biocontrol fungi were investigated. These results provide some insights into the dynamic changes in the microbial community in the root system and the adaptability of C. fascicularis to the cultivation environment.

2. Materials and Methods

2.1. Site Description and Sample Processing

The sampling sites were located in Hekou County of Yunnan Province, China, where C. fascicularis is most viable in the wild (22°40′17.69″ N, 103°56′30.3″ E, Supplementary Figure S1, Supplementary Table S1). C. fascicularis has been artificially cultivated three times since it was discovered. All the C. fascicularis samples from the same planting years were planted in the same sample sites, labeled Age_3, Age_5, and Age_7. The linear distance of each plot was more than two kilometers, and the rhizosphere microbes of C. fascicularis of different ages were compared horizontally. Three healthy C. fascicularis roots and the rhizosphere soil were collected from each sample site on 21 August 2023.

For each individual, the sampling process is as follows: After weeds and topsoil were eradicated, the rhizosphere soil samples were collected for chemical property determination from two opposite root-rich areas of C. fascicularis, which were 10~30 cm in depth. After removal of loose soil at the root surface and gravel in the root crevices, the soil at the root surface was gently collected in a 2 mL frozen tube for high-throughput sequencing. After the rhizosphere soil was collected, the fine roots of C. fascicularis were collected and were cut with scissors, sterilized with 75% ethanol, and placed in a new 2 mL frozen tube. Three individuals as three biological replicates from each sampling site were collected. All samples were stored on dry ice. The frozen tubes were transported to the laboratory and stored at −80 °C. In addition, fine roots were cleaned and ultrasonically treated for 10 min using ultrasonic bath cleaning (Branson, CT, USA) [6]. The roots were surface sterilized in 0.5% hypochlorite (NaClO) for 5 min, in 75% ethanol (CH₃CH₂OH) for 5 min, and then washed with sterile water. The final washing water was inoculated into trypsin soybean agar (TSA) medium and potato glucose agar (PDA) medium to verify the success of surface sterilization.

2.2. Soil and Root Chemical Properties

The loose soil around the rhizospheres of the 9 individual C. fascicularis plants from the above 3 sample sites (Age_3, Age_5, and Age_7) were taken back to the laboratory. After a week of natural drying, the soil was filtered with a 0.2 mm sieve, and then the soil organic carbon, total nitrogen, total phosphorus, and pH were measured. Briefly, the organic carbon of the soil and roots was measured using the potassium dichromate (K₂Cr₂O₇)–sulfuric acid (H₂SO₄) volumetric method [21]. Total nitrogen transformed as NH₄⁺ was analyzed using a Smartchem200 after digestion by concentrated hydrogen peroxide (H_eO₂)–sulfuric acid (H₂SO₄), and the total phosphorus content was also measured using a SmartChem200 automatic chemical analyzer (AMS-Westco, Italy) [22,23]. The pH value of the soil was measured with a pHS-2F pH meter (INESA Scientific Instrument Co., Ltd., Shanghai, China).

2.3. DNA Isolation and Sequencing

For high-throughput sequencing, total DNA of 200 mg of sterilized fine roots was extracted using the CTAB method for each root sample, and 0.5 g of soil was extracted with a commercial DNA extraction kit for each rhizosphere soil sample (FastDNA^® SPIN Kit for Soil, MpBio). DNA quality was detected using 0.8% agarose gel electrophoresis, and extracted DNA was diluted to a concentration of 1 ng/μL. The diluted DNA was used as a template for PCR amplification. Then, 16S rRNA genes and ITS fragments were amplified using specific primers (799F: 5′-AACMGGATTAGATACCCKG-3′ and 1193R: 5′-ACGTCATCCCCACCTTCC-3′ for 16S rRNA; ITS1-F: 5′-CTTGGTCATTTAGAGGAAGTAA-3′ and ITS2: 5′-GCTGCGTTCTTCATCGATGC-3′ for ITS) [24] tagged with a barcode. PCR amplification was conducted in a total reaction volume of 25 μL containing 5 μL of 5× reaction buffer, 5× GC buffer 5 μL, dNTP (2.5 mM) 2 μL, forward primer (10 μM) 1 μL, reverse primer (10 μM) 1 μL, DNA Template (1 ng/μL) 2 μL, ddH₂O 8.75 μL, and Q5 DNA Polymerase (100 units) 0.25 μL. The thermal cycling consisted of initial denaturation at 98 °C for 2 min, 25 cycles of denaturation at 98 °C for 15 s, annealing at 55 °C for 30 s, extension at 72 °C for 30 s, and final extension at 72 °C for 5 min. Then, library construction and sequencing were performed using the Illumina MiSeq PE250 platform by Personalbio, Inc. (Shanghai, China). Raw sequences were deposited in the Sequence Read Archive under Bioproject PRJNA1028152.

2.4. Bioinformatic Analysis

According to the official tutorial (https://docs.qiime2.org/2019.4/tutorials/, accessed on 23 September 2023), we performed a microbiome bioinformatics analysis using QIIME 2 with slight modification [25]. Briefly, the demux plugin was used to demultiplex the raw sequence data and then we used the Cutadapt plugin for primer cutting [26]. Next, the DADA2 plugin was used for quality filtering, denoising, merging, and chimera removal of the sequences [27]. Non-singleton amplicon sequence variants (ASVs) were aligned with mafft [28] and used to construct a phylogeny with fasttree2 [29]. Taxonomy was assigned to ASVs using the classify-sklearn naive Bayes taxonomy classifier in the feature-classifier plugin [30] against SILVA_138_1 for bacteria and UNITE_9 for fungi.

2.5. Data and Statistical Analyses

All reads were obtained from 18 samples (3 rhizosphere soils, 3 root samples × 3 cultivation years). We then subsampled the remaining 26,182 bacterial reads and 81,025 fungal reads (minimized read sample) using MOTHUR v1.35.1 “sub.sample”. All data were analyzed according to data descriptions based on subsampled data. The rarefaction curve for samples was calculated using the R v3.4.3 “vegan” package. Nonmetric multidimensional scaling (NMDS) based on Bray–Curtis dissimilarity was performed using the R v3.4.3 “vegan” package to investigate the differences among different cultivation years [31]. PERMANOVA (permutational multivariate analysis of variance) was used to test the statistically significant differences based on ASV richness using the R “vegan” package [31]. Alpha diversity was calculated using the R “vegan” package [31]. The Kruskal–Wallis test (for three groups) was performed using the R v3.4.3 “stats” package to investigate the significant differences among groups [32].

3. Results

3.1. Soil Chemical Properties

In order to study the chemical properties of the rhizosphere soil of C. fascicularis, the total carbon, total nitrogen, total phosphorus, and pH of the rhizosphere soil were measured and compared among different cultivation years (Figure 1). The results show that there were no significant differences in total carbon, total phosphorus, and pH values among the cultivation years (p > 0.05). The total nitrogen content of the rhizosphere soil at 7 years was significantly higher than that at 3 years (p < 0.05). The total carbon changed little in each year. The total nitrogen and total phosphorus increased, and the pH decreased gradually with the increase in cultivation years.

3.2. Differentiation of C. fascicularis Endophytes and Rhizosphere Soil Microbial Communities

In total, 9 rhizosphere soils and 9 root samples of fungi and bacterial communities were sequenced. All samples were first subsampled to 26,182 reads for bacteria and 81,025 reads for fungi, i.e., the minimized read sample. The rarefaction curve approached saturation at the minimized number of reads (Supplementary Figure S2). The alpha diversity indexes of rhizosphere soil were significantly higher than those of root endophytes (Figure 2). Additionally, there were significant differentiations between the rhizosphere soil and root endophytes of C. fascicularis according to NMDS (p = 0.001, PERMANOVA, Figure 2).

For fungal microbial community compositions, Ascomycota and Basidiomycota were the dominant phyla in both rhizosphere soil and roots. Ascomycota and Basidiomycota were more abundant in roots compared with soil. Melanconiella, Lunulospora, Dactylonectria, Atractiella, Gymnopilus, and Fusarium were the dominant genera in roots and Archaeorhizomyces, Trichoderma, Staphylotrichum, Melanconiella, Fusarium, and Penicillium were the dominant genera in soil (Figure 3).

For bacterial microbial community compositions, Proteobacteria and Actinobacteriota were the dominant phyla in both rhizosphere soil and roots. Actinobacteriota was more abundant in roots than in soil, at 46.79% and 12.67%, respectively (Figure 3). Acidothermus, Bradyrhizobium, Phenylobacterium, and Rhodomicrobium were the dominant genera in roots and Acidothermus, Haliangium, RCP2-54, Ellin6067, and KF-JG30-C25 were the dominant genera in rhizosphere soil (Figure 3).

3.3. Variation in Microbial Communities in Rhizosphere Soil and Roots of Camellia for Different Cultivation Years

To explore the variation in microbial communities in the soil and root samples, the alpha and beta diversities were analyzed. In general, there was no significant difference in the alpha and beta diversities between endophytes and rhizosphere soil for different cultivation years (Figure 4 and Figure 5). After artificial cultivation, the alpha diversity index of endophytic bacteria and rhizosphere soil fungi increased first and then decreased. However, the soil bacterial alpha diversity index decreased first and then increased. There was no significant difference in alpha diversity among different cultivation years. In terms of beta diversity, the microbial communities for different cultivation years were different, but there was no significant difference. Overall, we can infer that the microbial community changed during the 3- to 7-year artificial cultivation, but the difference is not statistically significant.

3.4. Variation in Main Pathogenic and Biocontrol Agents in Camellia with Different Cultivation Years

To explore changes in the main functional microbes, we analyzed the changes in pathogenic microbes and biocontrol agents in the Top 50 dominant genera compared with C. sinensis, as reported in the literature [15,16,17,18,19,20].

A total of 6 genera of PGPR were found in the root samples, i.e., Enterobacter, Pseudomonas, Bacillus, Streptomyces, Paenibacillus, and Burkholderia (Figure 6). Only 2 genera of PGPR were found in the rhizosphere soil samples, i.e., Burkholderia and Bacillus. However, their abundances were not significantly different in different cultivation years. In addition, there was no significant difference in the abundance of beneficial bacteria between soil samples and root samples at different cultivation times.

Fusarium, widely known as pathogenic fungi, had more abundance in 5-year rhizosphere soil compared with the 7-year samples (Figure 7). Three other genera, Trichoderma, Penicillium, and Aspergillus, reported as biocontrol agents in C. sinensis, were found in root and soil samples (Figure 7). The Trichoderma and Penicillium in the rhizosphere soil of the 5-year samples had more abundance than that in the 3-year samples. The abundance of the Aspergilluis in the 3-year root samples was higher than that in the 7-year samples.

4. Discussion

Camellia fascicularis is a PSESP, and one of the conservation measures used for such plants is the experimental translocation of threatened species into multiple locations within and outside their known range [33]. It is difficult to assess the environmental fitness of C. fascicularis because of a PSESP’s narrow distribution, even if cultivated in the same county. Similar to invasive plants, C. fascicularis inevitably accumulates local microbes after artificial cultivation [4] and also recruits the composition of root microbes mediated by root exudates [6,34]. Therefore, the structure of the microbial community in the root system of C. fascicularis in different years of artificial cultivation, the accumulation of pathogens in soil and roots, and the trade-off between pathogens and biocontrol agents are the issues discussed in this paper.

We investigated three C. fascicularis plots with different years of cultivation (labeled Age_3, Age_5, and Age_7) and compared the changes in the microbial communities in the three plots horizontally. The results showed that the composition of rhizosphere soil and root endophytes were significantly different. In agreement with most of the literature, the results show that the endophytes were not simply random or changed according to the rhizosphere soil microbial pool gradient, but were actively recruited [34,35]. The composition of soil microorganisms was consistent with previous reports. Proteobacteria, Acidobacteria, and Actinobacteria were the main bacterial taxa in the soil, while Ascomycota and Basidiomycota were the main fungal taxa (Figure 3) [5]. Interestingly, more Actinobacteriota were observed in root endophytes; this may be a useful source of several bioactive compounds with plant-growth-assisting (IAA production, ACC deaminase activity), antifungal, and antibacterial activity (Figure 3) [36]. However, the abundance of Proteobacteria in the roots was lower than that in the rhizosphere, which was different from that in the literature [5]; this may be related to the abundance of Actinobacteriota in the roots. Firmicutes were more abundant in the roots and Bacteroidota were more abundant in the rhizosphere soil, but, overall, their total abundance was less than 1% in this study (Supplementary Table S2).

In terms of fungi, there were more Ascomycota, Melanconiella (opportunistic pathogens and endophytic fungi) [37], and Penicillum (biocontrol agents) in the roots and more Trichoderma, known to be biocontrol agents, in the soil [38]. The abundances of Glomeromycota in the roots and rhizosphere soil were 0.036% and 0.287%, respectively. The soil in cultivated land is acidic (Figure 1) and the growth of C. sinensis in acidic soils depends heavily on AMF [13]. As the phylum of AMF fungi, Glomeromycota shows a symbiosis between Camellia spp. and mycorrhizal fungi, including C. japonica, C. sinensis, and C. oleifera [39]. However, when we compared the genera reported as AMF, we did not find the genera of mycorrhiza in our high-throughput sequencing [13]. The absence of AMF may be related to the unfitness of the ITS1 region for AMF amplification. C. fascicularis-AMF symbiosis also needs to be further studied. The genus Fusarium, known as pathogenic fungi of C. sinensis, was found in the soil and roots, which indicated that more local pathogenic fungi accumulated after artificial cultivation [20]. The above results indicate a trade-off between beneficial and harmful microbes. In addition, because the pathogenic microbes of C. fascicularis have not been studied systematically, the genera of common pathogenic microbes of C. sinensis were used in this study. Except for a very small amount of Colletotrichum fungi (outside the top 50 genera), the other C. sinensis pathogenic fungi, such as Pestalotiopsis, Exobasidium, Botryodiplodia, Macrophoma, Ustulina, and Sphaerotheca, were not found. Colletotrichum, as a fast-growing culturable fungus, is found more frequently in the literature for some culturing methods, but its abundance is lower in the high-throughput results, which also shows the complementarity between high-throughput and culturing methods [40]. It remains to be confirmed whether the other pathogens of C. sinensis can successfully infect C. fascicularis or if they accumulate in the root system after years of artificial cultivation.

After 3-year, 5-year, and 7-year artificial cultivation of C. fascicularis, there was no significant difference in the alpha and beta diversity of the root microbial community structure of C. fascicularis (Figure 4 and Figure 5). However, the change in rhizosphere soil microbes was slightly larger than that of root endophytes (Figure 4 and Figure 5), which once again shows that root endophytes were recruited from the rhizosphere [34]. According to the chemical composition of the rhizosphere soil, the total nitrogen of the rhizosphere soil after 7 years was significantly higher than that after 3 years (Figure 1). In addition, there were no significant differences in total carbon, total phosphorus, and pH among C. fascicularis with 3-, 5-, and 7-year cultivation. However, the effects of total nitrogen, total phosphorus, and pH on the microbial community were significant (Supplementary Figure S3). In addition, although the total nitrogen in rhizosphere soil has an increasing trend, the leaching of nitrogen and soil acidification still need long-term attention and investigation [34]. With the increase in years and nitrogen leaching, the soil pH may continue to decrease, which will lead to changes in the soil microbial structure and function [41]. Long-term follow-up investigation is therefore required.

In order to further determine the dynamic changes in the main beneficial and harmful microbes in C. fascicularis, the beneficial and harmful microbes of C. sinensis were analyzed. The results show more PGPR in root endophytic bacteria than in rhizosphere soil, but there was no significant difference in PGPR abundance between different cultivation years. These results suggest that more PGPR bacteria are endogenously recruited by the root system to promote plant host growth and improve resistance directly [42]. Meanwhile, Trichoderma, Penicillium, and Aspergillus were also found in the rhizosphere of C. fascicularis as biocontrol agents [38]. Aspergillus in the roots of C. fascicularis was significantly higher at 3 years than at 7 years, and the other biocontrol bacteria showed no significant difference for different cultivation years. Compared with the endophytic fungi, the biocontrol fungi Trichoderma and Penicillium in the rhizosphere soil cultivated for 5 years were significantly higher than those in the rhizosphere soil for 3 years. However, there was no significant difference between 7-year cultivation and 3- and 5-year cultivation. These results also suggest that rhizosphere soil is the first to be affected during cultivation, while endophytes will be affected over a longer period of time. The fungi in the roots and rhizosphere soil showed some differences among different cultivation years, but bacteria showed no significant difference in terms of soil, rhizosphere, and different cultivation years. These results reflect the fact that fungi are more susceptible to environmental influences [7]. Finally, we found more Fusarium pathogens in soil and roots, but no other common pathogens were found in the top 50 genera (Supplementary Table S3) [20]. This indicates that the accumulation of pathogenic fungi had begun after years of artificial cultivation, but the accumulation of soil-borne disease pathogenic fungi was still at a low level due to the existence of biocontrol agents and actinomycetes.

5. Conclusions

In order to study the changes in the soil chemistry and root microbial community structure of the PSESP C. fascicularis after artificial cultivation, three different artificial planting years’ plots were selected (labeled Age_3, Age_5 and Age_7); the soil chemical properties and the microbial community structures in root and rhizosphere soils were also compared horizontally. The results show that changes in the soil chemical properties and microbial community structure and the species and abundance of useful and harmful microbes in C. fascicularis were not significant among three plots. These results are helpful for understanding the adaptability of C. fascicularis to artificial cultivation, and it is necessary to investigate the microbes of C. fascicularis for a long period.