Effects of elevated atmospheric CO₂ on rhizosphere microbial community of Pinus densiflora (Korean red pine)

Abstract

The concentration of atmospheric CO₂ is increasing largely owing to human activities. Although it is well established that elevated CO₂ (eCO₂) stimulates plant growth and primary productivity, the effect of eCO₂ on soil microbial communities remains poorly understood. In this study, we aimed to examine the effects of eCO₂ on the taxonomical diversity, composition, and structure of rhizosphere microbial communities of Korean red pine (Pinus densiflora) through next-generation sequencing (NGS) of 16S rRNA genes. Three bacterial phyla, Proteobacteria, Acidobacteria, and Actinobacteria, were found to be dominant in all samples. Species richness estimates (Chao1 and ACE) and diversity indices (Shannon and Simpson) for the three sampling chambers were the highest from the eCO₂ (approximately 1.4× ambient, ≈ 560 ppm). These findings suggested that elevated atmospheric CO₂ affected the bacterial community composition in the rhizosphere of Korean red pine (Pinus densiflora).

Keywords:

• Climate change
• elevated CO₂
• microbial diversity
• open-top chamber
• Pinus densiflora
• rhizosphere microbes

Introduction

The effect of climate change and global warming on the natural forest ecosystem as well as the trees in the ecosystem are very important in relation to global climatic changes, as they play a key role in the global carbon balance and carbon sequestration (Ncipha and Sivakumar 2019). Carbon dioxide (CO₂) concentration started at around 280 parts per million (ppm) in pre-industrial times, but it now exceeds 400 ppm and is expected to reach 600–800 ppm by the end of the century (Quirk et al. 2019).

To understand the physiological response and determine the future consequences of the increasing atmospheric CO₂ concentration on trees, long-term field experiments are necessary to improve our understanding of the responses of trees (Morin et al. 2018). Several trees, including Pinus radiata (Tissue et al. 2001), P. ponderosa (Pushnik et al. 1999), Norway spruce (Bader et al. 2016), populus (Yadav et al. 2019), P. sylvestris (Jach and Ceulemans 1999), Picea sitchensis (Laitat and Boussard 1995), and Picea abies (Janouš et al. 2000), have been shown to control CO₂ concentrations in open-top chambers (OTCs), which provide moderately precise environmental controls in relatively natural, field-like conditions.

It is well documented that elevated CO₂ (eCO₂) induces an initial stimulation of plant growth (Jifon and Wolfe 2005), and studies on soil microbial community around plant roots under eCO₂ have been performed with crop plants, as their rhizosphere contains a diverse and complex microbial structure and a community critical for the health of plants and the processing of soil organic matters (OMs) (Nie et al. 2015; Wang et al. 2017). Despite the abundance and the importance of soil microorganisms for key forest ecosystem functions, the effects of eCO₂ on rhizosphere microbial communities in trees have not been well characterized.

Korean red pine (P. densiflora) is one of the most economically and culturally important tree species in Korea and widely distributed throughout Korea, Japan, and northeastern China (Kang et al. 2015). However, the growth and habitats of conifer trees, including pine tree, have been reduced owing to climate change and global warming (Battles et al. 2008; Jo et al. 2019). It is well known that beneficial plant-associated microorganisms promote plant growth and enhance plant resistance to various environmental stresses, including eCO₂, drought, warming, diseases, and insects (Lau et al. 2017; Yadav et al. 2018). The symbiotic relationship between fungi and pine roots has been well studied (Dhyani et al. 2019), but there is limited information available regarding soil microbes in the rhizospheres of Korean red pine. Therefore, the use of culture-independent molecular techniques has led to a new understanding of bacterial diversity, as improvements in sequencing technology have allowed for more robust estimates of microbial diversity in natural environments.

In this study, we used 16S rRNA gene-based pyrosequencing technique to investigate changes in soil microbial community structure under eCO₂ in an OTC experiment, which was established in South Korea. Korean pine trees were exposed to different concentrations of CO₂ in OTCs for 9 years. The objective of this study was to evaluate the rhizosphere microbial diversity of Korean red pine and to examine how CO₂ affects changes in microbial community.

Materials and methods

OTC system

The OTC system was established for long-term studies to monitor the effect of climate change on Korean red pine and its community in Korea (37°15′N, 136°57′E; 49 m above sea level (a.s.l.)). The OTC facility consisted of three decagon chambers (10 m in diameter by 10 m in height) with controlled gas concentration. For this experiments, seedlings of 1-year old were planted on each OTC and investigations started 4 years after planting. The experiment consisted of three treatments: two eCO₂ levels (approximately 1.4× (560 ppm) and 1.8× (680 ppm) ambient CO₂) and one control chamber (ambient CO₂, 380 ppm). Differences in temperature and relative humidity between the three OTC chambers were not detected.

Growth measurements

The heights and root diameters of Korean red pine were annually measured at the end of the growing season after the trees were planted.

Chemical analyses of plant leaf and soil

The collected leaves samples were freeze-dried and ground, and carbon and nitrogen concentrations were determined by elemental analyzers (Thermo Scientific, Flash 2000, Rodano, Italy). Phosphorus concentration was pulverized sample after wet digestion (Kalra and Maynard, 1991), and then it was analyzed using ICP (Perkin Elmer Optima 5300, Waltham, MA). Three replicates of rhizosphere soils and 500 g of fresh sunlit leaves of pine trees were collected from the three Korean red pines for each treatment. The collected soil samples were filtered to 2 mm after air-dried, and physicochemical analysis was performed according to the methods of Soil and Plant Analysis I (National Institute of Forest Science 2014a) and II (National Institute of Forest Science 2014b) by National Institute of Forest Science. Soil acidity and electrical conductivity (EC) were measured using pH and conductivity meter after shaking with distilled water at a ratio of soil and water of 1:5 (s). OM content in the soil was measured by oxidizing OM with potassium dichromate to measure organic carbon content, and multiplied by the conversion factor (1.724) to quantify the OM content (Tyurin method). Total nitrogen (TN) was completely decomposed by adding concentrated sulfuric acid and catalyst to the soil sample, collected in boric acid after Kjeldahl distillation, and then it was titrated with NH₄⁺ using 0.1 N sulfuric acid to quantify TN (%) in the soil. Available phosophate (mg/kg) was analyzed using a UV spectrophotometer after leaching phosphoric acid from soil by the Bray I method and color-developing with ascorbic acid. Cation exchange capacity was measured by adsorbing the soil surface with NH₄⁺ ions using ammonium acetate, and then titrating NH₄⁺ through Kjeldahl distillation. Cations (K+, Na+, Ca²⁺, Mg²⁺) were analyzed for each concentration by measuring the solution leached with ammonium acetate with an atomic absorption spectrophotometer (AAS).

PCR amplification and Illumina MiSeq sequencing

Three replicates of rhizosphere soil DNA from Korean red pine in the three different CO₂ concentration treatment chambers were extracted using a Power Soil DNA extraction kit (Mobio, Carlsbad, CA). Pyrosequencing was conducted in accordance with a previous research (Lee and Eom 2016). PCR reaction was carried out using primers targeting the V1–V3 regions of the 16S rRNA gene from the extracted soil DNA. The primers used for bacterial amplification were 27 F (5 (CCTATCCCCTGTGT-GCCTTGGCAGTC-TCAG-AC-GAGTTTGATCMTGG-CTCAG-3CAG-33T518R (5′-CCATCTCATCCCTG-CGTGTCTCCGAC-TCAG-X-AC-WTTACCGCGGCT-GCTGG-3′). Sequencing was performed at Chun lab, Inc. (Seoul, Korea) using a 454 GS FLX titanium next-generation sequencing (NGS) system (Roche, Branford, CT) according to the manufacturer’s instructions and previously described procedures (Chun et al. 2010).

Sequencing data analysis

Sequencing data were analyzed as previously described (Chun et al. 2010; Lee and Eom 2016). For NGS analysis, the obtained reads were subjected to quality check and trimming of low-quality scores (defined as average scores of <25) by Trimmomatic version 0.32 (Bolger et al. 2014). The complete hierarchical taxonomic classification of each read was assessed using a BLASTN search against the EzTaxon-e database (http://eztaxon-e.ezbiocloud.net) containing 16S rRNA gene sequences of type strains that had valid published names and representative species-level phylotypes (Kim et al. 2012). The richness and diversity of pine rhizosphere microbial communities were determined by Chao1 estimation and Shannon diversity index at the 3% distance. The overall phylogenetic distance among the treatments was estimated using Fast UniFrac (Hamady et al. 2010). To compare operational taxonomic units (OTUs) between samples, shared OTUs were obtained with the CL community program’s Taxon XOR by CD-HIT analysis (Chunlab Inc., Seoul, South Korea).

Results

The annual growth in height and diameter of Korean red pine is presented in Figure 1. The results showed that plant growth tend to increase with increasing CO₂ concentration owing to the CO₂ fertilization effect caused by high concentrations of CO₂. However, the difference in growth was not significant.

Figure 1. Cumulative heights and root diameters of Korean red pine from three different CO₂ concentration treatment chambers. OTC1: Elevated CO₂ level as 1.4x (approximately 560 ppm), OTC2: elevated CO₂ level as 1.8x (approximately 680 ppm), and OTC3: ambient CO₂ level (approximately 380 ppm).

NGS of 27 rhizosphere soil samples from Korean red pine resulted in approximately over 13,569 reads per sample, representing 40 phyla, 150 classes, and 500 genera of bacteria and archaea, as well as 2045–2874 OTUs at a similarity level of 97%. The numbers of observed OTUs from the rhizosphere of pine trees were significantly different among different CO₂ concentrations in the OTC chambers. The rarefraction curves calculated with QIIME pipeline at 97% similarity also showed different OTU richness patterns for all three chambers (Figure 2).

Figure 2. Rarefaction curves for bacterial operational taxonomic units (OTUs; 97% sequence similarity) in each sampling chamber associated with the rhizosphere of pine tree. In the rarefaction curves, the number of OTUs increased with the number of sequencing reads. OTC1: elevated CO₂ level as 1.4x (approximately 560 ppm), OTC2: elevated CO₂ level as 1.8x (approximately 680 ppm), and OTC3: ambient CO₂ level (approximately 380 ppm).

OTU richness estimations, such as Chao 1, indicated that samples collected from control chamber OTC1 (ambient CO₂) contained the lowest number of bacteria. However, exposure to eCO₂ altered the microbial populations and diversity. Comprehensive OTU richness analysis results (Table 1 and Figure 2) revealed that the bacterial community of OTC2 (CO₂ × 1.4) was the most diverse. Rarefaction analysis of the OTUs also indicated that bacterial diversity and richness significantly increased as CO₂ concentration increased (p < .01). In particular, bacterial diversity and richness from OTC2 (approximately 1.4× ambient, ≈560 ppm) were the highest.

Table 1. Comparison of the estimated OTU richness and diversity indexes in the rhizosphere of Korean red pine according to CO₂ concentration.

Sample	Valid reads	OTUs	Ace	Chao1	Jackknife	NPShannon	Shannon	Simpson	Goods Lib. coverage
OTC1	13,569	2045	3013.8	2730.3	2911.1	6.45	6.22	0.0112	91.8
OTC2	16,179	2874	3761.5	3500.0	3853.5	7.41	7.20	0.0018	92.2
OTC3	15,989	2524	3400.5	3122.6	3401.3	7.07	6.88	0.0032	93.2

Shannon index analysis revealed that the values of bacterial diversities in each chamber were 6.22 in OTC1, 7.20 in OTC2, and 6.88 in OTC3, which were consistent with the results of rarefaction analysis and the rank-abundance curves (Table 1).

Each bacterial 16S rRNA gene sequence was taxonomically assigned from the phylum level to the species level using the Ribosomal Database Project Classifier. The proportion of unclassified bacteria was no more than 0.1%. A total of 44 phyla were identified in all three chambers. Particularly, three bacterial phyla, namely Proteobacteria, Acidobacteria, and Actinobacteria, were the most dominant in all three chambers (Figure 3). The three dominant phyla accounted for 69.1%, whereas other phyla accounted for 30.9%. The relative abundance was more than 37.2% for Proteobacteria, 17.6% for Acidobacteria, and 14.3% for Actinobacteria. The following phyla were also observed: Planctomycetes (5.5%), Chloroflexi (5.3%), Bacteroidetes (4.8%), Verrucomicrobia (4.6%), Gemmatimonadetes (1.8%), Firmicutes (1.2%), Cyanobacteria (1.3%), Chlamydiae (2.5%), Nitrospirae (1.8%), Armatimonadetes (1.2%), and others (1.7%), with less than 1% composition for each phylum (Figure 4).

Figure 3. Similarity of phylogenetic diversity at the phylum level (97% sequence similarity) for bacterial communities. The hierarchical relationship was obtained via the unweighted pair group method with arithmetic mean (UPGMA) clustering dendrogram. OTC1: elevated CO₂ level as 1.4x (approximately 560 ppm), OTC2: elevated CO₂ level as 1.8x (approximately 680 ppm), and OTC3: ambient CO₂ level (approximately 380 ppm).

Figure 4. Relative distribution (%) of the main bacteria taxa detected in the rhizosphere of Korean red pine tree according to CO₂ concentration. OTC1: elevated CO₂ level as 1.4x (approximately 560 ppm), OTC2: elevated CO₂ level as 1.8x (approximately 680 ppm), and OTC3: ambient CO₂ level (approximately 380 ppm).

We compared the constituent taxa of rhizosphere soil bacterial communities and their rank orders among all chambers. At the phylum level, Proteobacteria was the most abundant phyla in the rhizosphere soil of pine tree. The proportion of Proteobacteria, Gemmatimonadetes, and Firmicutes significantly increased with eCO₂ as follows: Proteobacteria: 36.6% in OTC1, 37.2% in OTC2, and 38.9% in OTC3; Gemmatimonadetes: 0.9% in OTC1, 2.2% in OTC2, and 2.2% in OTC3; Firmicutes: 0.5% in OTC1, 1.2% in OTC2, and 1.8% in OTC3.

At the class level, Alpha-proteobacteria (18.7%), Beta-proteobacteria (10.3%), Actinobacteria (9.7%), Solibacteres (4.7%), Verrucomicrobiae (4.3%), and Gammaproteobacteria (4.0%) were observed. Interestingly, the proportion of Actinobacteria significantly decreased as CO₂ concentration increased. The proportions were 16% in OTC1, 6% in OTC2, and 7% in OTC3.

In the family level, the proportions of Bradyrhizobiaceae (18.3%), Acidobacteriaceae (11.3%), and Planctomycetaceae (11%) significantly increased in the eCO₂. The proportions were was 4.2% in OTC1, 6.2% in OTC2, and 7.9. The proportion of Acidobacteriaceae (11.3%) also increased as CO₂ concentration increased.

In the genus level, the proportion of Acidobacteria significantly increased with increasing CO₂: 11% in OTC1, 19.8% in OTC2, and 21.9% in OTC3. However, those of Actinobacteria, Cyanobacteria, and Bacteroidetes decreased as follows: Actinobacteria: 20.1% in OTC1, 10.6% in OTC2, and 12.1% in OTC3; Cyanobacteria: 3.2% in OTC1, 0.3% in OTC2, and 0.3% in OTC3; Bacteroidetes: 7.8% in OTC1, 3.7% in OTC2, and 2.8% in OTC3.

Foliar chemical analysis revealed that the total organic carbon, nitrogen, and total phosphate content tended to decrease with increasing CO₂. The total organic carbon and phosphate content in the leaves tended to decrease with increasing CO₂, although the decrease was not significant (Table 2). Rhizosphere soil chemical analysis showed that soil acidity, phosphoric acid, and CEC significantly decreased in the CO₂-treated groups. Soil OM, potassium, sodium, and magnesium content were not significant among three treatments (Table 3).

Table 2. Chemical properties of Korean red pine leaves according to CO₂ concentration.

Sample	Total organic carbon (%)	Nitrogen (%)	Phosphate (ppm)
OTC1	45.44 ± 1.27 ns	1.30 ± 0.07 a	0.49 ± 0.05 ns
OTC2	43.18 ± 1.55 ns	1.14 ± 0.06 ab	0.39 ± 0.08 ns
OTC3	41.78 ± 1.07 ns	1.08 ± 0.02 b	0.42 ± 0.03 ns

Different lowercase letters indicate statistically significant difference at 5% level in columns. ns: not significant.

Table 3. Soil chemical properties in the rhizosphere of Korean red pine according to CO₂ concentration.

Sample	pH	OM (%)	TN (%)	TP (mg kg^−1)	CEC (cmol kg^−1)	K^+(mg kg^−1)	Na^+(mg kg^−1)	Ca^2+ (mg kg^−1)	Mg^2+(mg kg^−1)	EC (dS m^−1)
OTC1	6.73 ± 0.03 a	0.35 ± 0.04 ns	0.01 ± 0.00 a	28.30 ± 1.01 a	1.98 ± 0.04 a	0.08 ± 0.00 ns	0.06 ± 0.00 ns	2.81 ± 0.01 a	0.48 ± 0.03 ns	0.07 ± 0.00 a
OTC2	6.07 ± 0.03 b	0.36 ± 0.05 ns	0.09 ± 0.00 a	16.13 ± 2.21 b	1.27 ± 0.15 b	0.08 ± 0.01 ns	0.05 ± 0.00 ns	1.60 ± 0.13 b	0.44 ± 0.05 ns	0.05 ± 0.00 b
OTC3	5.90 ± 0.15 b	0.39 ± 0.06 ns	0.11 ± 0.00 b	18.23 ± 2.91 b	1.56 ± 0.21 ab	0.07 ± 0.00 ns	0.05 ± 0.00 ns	1.78 ± 0.18 b	0.42 ± 0.01 ns	0.06 ± 0.00 b

Different lowercase letters indicate statistically significant difference at 5% level in columns. ns: not significant.

Discussion

We evaluated microbial communities in the rhizosphere of Korean red pine and compared them among different concentrations of CO₂ in OTCs with Illumina MiSeq high-throughput sequencing. This was the first study to describe microbial community composition in the rhizosphere of Korean red pine. The microbial community plays an important role in the soil biochemical cycle. In addition, many environmental factors, such as soil OM chemistry, plant species, soil pH, and environmental factors, can affect rhizosphere microbial diversity. In the case of rhizosphere, the community has a very close relationship with plant growth and health, as the root exudate composition can affect rhizospheric microbial communities (An et al. 2020). In the phylum level, three bacterial phyla, including Proteobacteria, Acidobacteria, and Actinobacteria, accounted for a high proportion of pine rhizosphere soil. In particular, Proteobacteria occupied the highest proportions in the rhizosphere. In general, Proteobacteria is a major group (phylum) of soil bacteria, and its abundance in the soil promotes various substrates to thrive and thus plays a very important role in nitrogen fixation, substrate degradation, and biochemical cycles, especially in soil ecosystems (Malisorn et al. 2020). Acidobacteria are also widely distributed and abundant in soils, but their ecological roles are poorly understood (He et al. 2012). Most phylotypes were detected at both ambient CO₂ and eCO₂, with only a few detected at only ambient CO₂ or only eCO₂. At the phylum level, the number of OTUs was 4740 (36.6%) in OTC1, 5822 (36.2%) in OTC2, and 6194 (38.9%) in OTC3 for Proteobacteria, a phylum with the highest number of detectable OTUs, followed by Acidobacteria (17.6%) and Actinobacteria (14.3%). Firmicutes and Bacteroidetes are involved in fermentation. However, in samples from the eCO₂-treated groups, the proportion of Firmicutes and Bacteroides was only 1.2 and 4.8%, respectively.

Members of the Alpha, Beta, and Gamma sub-phyla of Proteobacteria are considered eutrophs, and they are more prevalent where resource availability is high, such as in rhizosphere soils. Generally, elevated atmospheric CO₂ stimulates the flow of organic C into the soil system (Kuzyakov et al. 2019). In addition, CO₂ increases root production and exudation. Moreover, eCO₂ stimulates photosynthesis, leading to increased carbon uptake and assimilation, thereby increasing plant growth and microbial biomass (Prior et al. 2011). There is little evidence that atmospheric CO₂ enrichment will increase total soil OM content because greater C flow into soil stimulates the soil food web, often leading to equivalent increases in soil CO₂ efflux (Pritchard 2011).

Bradyrhizobiaceae (18.3%) is a family of Rhizobiales order in the Alphaproteobacteria class. It includes plant-associated bacteria, such as Bradyrhizobium, a genus of rhizobia that could fix nitrogen (Larimer et al. 2004). However, nitrogen was significantly reduced under eCO₂. These phenomena were consistent with reports from previous studies that when CO₂ concentration in the air increases, the TN content of the leaves decreases. Although three trees in the treatment groups were not sufficient to show significant differences, there was no decrease in productivity parameters, such as height and root collar diameter, due to nitrogen reduction. Growth is expected to decrease as the period of exposure to high-concentration CO₂ increases. Annual growth analysis in this study revealed that exposure to CO₂ initially had a significant effect on plant growth owing to the fertilizing effect of CO₂, but it is believed that the effect of high-concentration CO₂ will not last long. For this reason, it is expected that the diversity and abundance of microorganisms and various indices were the highest in OTC2 (approximately 1.4× ambient, ≈560 ppm).

Our current findings indicated that the composition and variation of microbial communities of endogenous bacteria or fungi in Korean red pine in different eCO₂ conditions. These studies will help elucidate the interactions between microorganisms and pine trees in a more comprehensive manner, overcome challenge associated with climate change, and promote health.

Conclusion

By comparing the characteristics of Korean red pine rhizosphere bacterial community among different concentrations of CO₂ by Illumina MiSeq sequencing, we showed that there were differences in the dominant rhizosphere bacterial taxonomic composition and community functions among the different concentrations of CO₂. Moreover, CO₂ concentrations higher than 560 ppm were shown to be beneficial for plant growth in the early stages, but damaging in the long term. The richness of the rhizosphere bacterial communities and the abundance of beneficial microbes, such as Acidobacteria, Gemmatimonadetes, and Firmicutes, increased as CO₂ concentration increased. Overall, our results revealed atmospheric CO₂ concentration as the main factor influencing bacterial community composition and diversity in the rhizosphere of the Korean red pine. In addition, our results suggested that the compositional structure of bacterial communities in the rhizosphere of pine tree could be determined by conditions associated with CO₂ in the air, as climate change due to increased atmospheric CO₂ affects physiological mechanisms, such as photosynthesis, and this usually leads to an increase in growth. Thus, our study confirmed a strong positive correlation between CO₂ concentration and composition of rhizosphere microbes, providing a strategy to cope with climate change, which causes destruction of the habitat of conifer trees, through improvement of soil bacterial communities.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

This research was funded by the National Institute of Forest Science and also supported by Kyungpook National University Research Fund of 2018.