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Article

Composition and Diversity of Soil Microbial Communities in Walnut Orchards at Different Altitudes in Southeastern Tibet

by
Ruyu Yan
1,2,
Ximei Zhao
1,2,
Penghui Li
2,3,
Zhuanyun Si
2,3,
Yang Gao
2,3 and
Jifu Li
1,2,*
1
College of Agriculture, Yangtze University, Jingzhou 434025, China
2
Key Laboratory of Crop Water Requirement and Regulation, Ministry of Agriculture and Rural Affairs, Xinxiang 453002, China
3
Farmland Irrigation Research Institute, Chinese Academy of Agricultural Science, Xinxiang 453002, China
*
Author to whom correspondence should be addressed.
Land 2023, 12(7), 1419; https://doi.org/10.3390/land12071419
Submission received: 15 June 2023 / Revised: 12 July 2023 / Accepted: 12 July 2023 / Published: 15 July 2023
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Abstract

:
To understand the distribution of the soil microbial community in natural walnut orchards at different altitude gradients (3000–3500 m) and to reveal the mechanism of the soil microbial activity in natural walnut orchards adapting to high-altitude environments, soil samples from four groups of natural walnut orchards in Gyaca County, southeast Tibet, were studied. Illumina MiSeq sequencing technology was used to analyze the community composition and diversity of soil bacteria and fungi and their responses to the altitudes. The alpha diversity results showed that the vertical distribution pattern of the fungal community was more obvious than that of the bacterial community and the bacterial community diversity first increased (~3364 m) and then decreased with altitude. The number of amplicon sequence variants (ASVs) in the soil bacterial community was significantly higher than that in the fungal community, but soil bacterial and fungal communities in walnut orchards at different altitudes exhibited both inheritance and uniqueness. At the phylum level, the dominant bacterial phyla at different altitudes were Actinobacteria, Acidobacteria, Proteobacteria, and Chloroflexi (relative abundances > 10.0% in each treatment). With the increase in altitude, the relative abundance of Actinobacteria increased gradually while that of Acidobacteria and Proteobacteria decreased gradually. The dominant fungal phyla were Ascomycota, Basidiomycota, and Mortierellomycota (relative abundances >5.0% in each treatment). With the increase in altitude, the relative abundance of Ascomycota increased significantly. At the genus level, the number of dominant bacteria and fungi in the soil decreased gradually with increased altitude and showed anisotropic distribution characteristics. The relative abundances of Actinobacteria among the bacterial phyla—and Olpidiomycota and Zoopagomycota among the fungal phyla—were positively correlated with the altitude (p < 0.05). Most dominant bacterial and fungal phyla were highly significantly (p < 0.01) or significantly (p < 0.05) negatively correlated with the altitude. Soil nitrogen and phosphorus availabilities are the main limiting factors of microbial community diversity. Therefore, altitude caused changes in soil physicochemical properties which directly or indirectly affected the composition and diversity of soil bacterial and fungal communities, and our study provides a theoretical basis for the altitudinal pattern and succession changes in soil microbial communities in the natural walnut orchards of southeast Tibet.

1. Introduction

Soil health is an important factor in maintaining the productivity and sustainable development of terrestrial ecosystems, especially microorganisms, as they are the most dynamic component of soil ecosystems and play an important role in soil formation, material transformation, energy flow, and other soil processes [1,2,3]. Bacteria and fungi are two main groups of soil microorganisms that are important links to achieve soil nutrient cycling and improve soil fertility. Beneficial microorganisms can create a good growth environment for host plants and form a synergistic relationship between the microorganisms and the plants. Therefore, the interaction between bacteria, fungi, and plants is of great significance for the ecological stability of the environment [4,5].
Walnut is a high-value, broad-leaved forest tree whose fruit (walnut kernel) is rich in protein and unsaturated fatty acids and is a major economic product [6]. According to their different origins, Chinese walnut varieties can be divided into the Xinjiang walnut, the North China mountain walnut, the Qin-ba mountain walnut, and the Tibetan mountain (plateau) walnut. At present, in-depth studies have been conducted on the breed improvement, nutritional characteristics, cultivation management, and soil fertility of the first three kinds of walnut, and the industrial chain is relatively complete [7,8,9,10]. However, attention to the walnut industry in the Tibetan Plateau is still relatively inadequate. The Tibetan Plateau walnut is a high-quality walnut that is cultivated without the application of chemical fertilizers and pesticides. Traditionally, fallen leaves and plant residues have been the main sources of nutrients, but the nutrient supply was not enough to meet the growth demands of the walnut tree. Therefore, soil fertility is the limiting factor for the high-quality and high-yield of the Tibetan Plateau walnut. In particular, the mineralization cycle of a large number of elements, such as nitrogen (N), phosphorus (P), and potassium (K), requires the active participation of microorganisms so that inactive nutrients can be converted into available nutrients for the absorption and utilization of walnut tree roots [5,11]. Plateau walnuts are widely distributed in the Tibetan region, with a suitable growing environment between 3000 and 4500 m above sea level. The microorganisms near the roots of walnut trees are closely attached to the rhizosphere soil particles and there is an adaptive coevolutionary relationship between these microorganisms and the roots [12]. Environmental factors such as temperature, rainfall, and solar radiation vary greatly at different elevations, which indirectly influences the distribution of the soil bacterial and fungal community diversity along the vertical gradient and ultimately affects the availability of soil nutrients. Yang et al. [13] found that the species of the soil bacterial community on the Qinghai–Tibet Plateau showed a single-peaked distribution along the altitude gradient but the dominant bacteria were Proteobacteria, Acidobacteria, and Actinomyces, which were not affected by altitude. The report of He et al. [14] in the Daiyun Mountain also showed that Proteobacteria, Acidobacteria (oligotrophic bacteria), and Actinomyces were the dominant bacterial groups at an altitude of 900–1500 m and that the soil total N, available N, and available P were the main abiotic factors driving the evolution of the dominant bacterial community composition and the diversity in forest soil at different altitudes. Research conducted by Nottingham et al. [12] on the distribution characteristics of microorganisms in the Andes Mountains showed that with the increase in altitude, the diversity of bacterial and fungal communities showed a downward trend. Cao et al. [15] sequentially analyzed the soil of the Shergyla Mountain in southeast Tibet and showed that Ascomycota and Basidiomycota were the dominant fungal groups in the soil. However, the Ascomycota was categorized as eutrophic taxa and the Basidiomycota was categorized as oligotrophic taxa [13,15]. At 3900 m, the diversity of the fungal community decreased significantly compared with that at 500 m. At present, studies on soil microorganisms using the altitude gradients on the Tibetan Plateau are mostly focused on terrestrial ecosystems such as grassland, forest, tjaele, and meadow ecosystems [15,16,17,18], while studies on the diversity of bacteria and fungi in agricultural land and economic woodland, especially in natural walnut orchards, are still relatively scarce and theoretical knowledge on the Tibetan Plateau of the microbial mechanism of nutrient cycling in the soil where the walnut has been cultivated for a long time is lacking.
Therefore, the soils of natural walnut orchards in the Tibetan Plateau were investigated to analyze and discuss the composition and influencing factors of the soil bacterial and fungal communities at different altitudes; thus, we tried to answer the following questions: (1) how the composition and diversity of the soil bacterial and fungal communities in natural walnut orchards in the Tibetan plateau changed with altitude gradient; (2) the effects of different soil nutrient factors on the soil bacterial and fungal community composition. Through this study, we hope to understand the elevation pattern of the soil bacterial and fungal communities and their driving factors in natural walnut orchards in the Tibetan Plateau and provide a theoretical basis for the composition of the soil bacterial and fungal communities and their driving mechanisms under different succession processes and stages.

2. Materials and Methods

2.1. Experimental Site Description

The experimental site is located in Gyaca county, Shannan city, in the southeastern Tibet Autonomous Region, China (29°14′35″ E, 92°33′17″ N), and in the middle reaches of the Brahmaputra River Valley. The site belongs to the plateau-temperate semi-arid monsoon climate zone, with an average annual temperature of 8.9 °C and an average annual rainfall of 493 mm. Gyaca County straddles the Gangdise Mountains, the Brahmaputra River plate junction zone, and the Himalayan plate, with a complex geological structure and soil-forming environment. The soil parent material mostly consists of metamorphic rocks such as killas, gneiss, phyllite, and loose Quaternary deposits [19]. The soil distribution in Gyaca County has typical mountain landform characteristics. The valley soil mostly consists of alluvial soil, the mountainside soil is grassland soil, and the mountaintop soil is meadow soil. The plateau walnut was distributed at different altitudes and was mainly found growing in natural ecological systems.

2.2. Experiment Design

The field experiment was conducted in July 2022 and four natural walnut orchards at different altitudes and which have not been disturbed by artificial factors (Table 1) were selected in the main walnut-producing area of Gyaca County, Shannan city, in the Tibet Autonomous Region. At an altitude of 3248 m, the walnut orchard (age of stand, ~20 yr) was artificially planted without other human interference, such as fertilization. The other three walnut orchards were naturally formed. To reduce the sampling errors, walnut trees with similar growth ages in the four orchards were selected for sample collection and a multi-point sampling method was used. One mixed sample (from the 0 to 40 cm soil layer) was collected from four directions near the drip line of the canopy in each walnut orchard, and the samples were repeated to obtain four replicates in each orchard for a total of 16 soil samples. The collected walnut orchard soil samples were placed into a Ziploc bag and brought back to the laboratory in a drikold box. After removing the debris from the soil samples, the samples were screened using a 2 mm mesh and stored in an ultra-low temperature refrigerator at −80 °C for the microbiological analysis.

2.3. Sample Determination

For the soil sample tests, the pH, the soil organic matter (SOM), and the total nitrogen (TN) in the soil were determined using a pH potentiometer, the potassium dichromate external heating method, and the Kjeldahl nitrogen determination method, respectively. The available nitrogen (available N), available phosphorus (available P), available potassium (available K), and the slowly available potassium (slowly available K) were determined using flow analysis, molybdenum-antimony resistance colorimetry, and flame spectrophotometry [20].
The total DNA of the soil samples (0.5 g) was extracted using the Omega Soil DNA Kit (Omega Biotechnology, USA) following the manual. The DNA length, concentration, and purity were measured using 1.2% agarose-gel electrophoresis and a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) [4]. The extracted soil DNA was stored at −80 °C for further bioanalysis.
An aliquot of the extracted soil DNA from each sample was used as the template for amplification. The V4 hypervariable regions of the bacterial 16S rRNA gene sequences and the ITS region of the fungal rRNA gene sequences were amplified [21]. Amplicon libraries were prepared using the following tagged bacterial and fungal universal primers: 515F and 806R for bacteria and ITS5 and ITS2 for fungi. The DNA samples were amplified individually using the pre-primer sequence 515F (5′-GTGYCAGCMGCCGCGGTAA-3′) and the posterior primer 806R (5′-GGACTACNVGGGTWTCTAAT-3′). ITS amplification of the fungus was performed using the pre-primer sequence ITS5 (5′-GGAAGTAAAAGTCGTAACAAGG-3′) and the post-primer ITS2 (5′-GCTGCGTTCTTCATCGATGC-3′) [5]. High-fidelity DNA polymerase and 25 µL reaction systems were used for the amplification of 16S and ITS, respectively. The polymerase chain reaction (PCR) amplification procedure was as follows: initial denaturation at 98 °C for 2 min, followed by 27 and 35 cycles of denaturation at 98 °C for 15 s for bacteria and fungi, respectively; annealing at 55 °C for 30 s with an extension at 72 °C for 30 s and a final extension at 72 °C for 5 min and a stop at 10 °C for preservation [22,23].
After amplification, the PCR products were recovered and purified using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, San Francisco, CA, USA) and quantified using a Quantus Fluorometer (Promega, Madison, WI, USA). The Illumina MiSeq sequencing platform was used to construct and sequence the library. The microbial diversity of bacteria and fungi was determined by Bioyigene Biotechnology Co., Ltd. (Wuhan, China).

2.4. Statistical Analysis

After the quality preliminary screening, the library and samples were divided according to index and barcode information, and the barcode sequence was removed for sequence denoising and clustering. The specific taxonomic composition of the walnut orchard species at different altitudes was displayed and the interactive display of community taxonomic composition was obtained using Krona software [22,23,24]. QIIME2 (2019.4) software was used to calculate the Chao1 index and the species index of soil microorganisms [13,25] using the undrawn amplicon sequence variant (ASV) table to represent the community species’ richness, and the Shannon index and the Simpson index were used [26,27,28] to represent the community species’ diversity. To determine which species were significantly affected among the treatments, the linear discriminant analysis effect size (LEfSe) algorithm was implemented [22]. All of the statistical analyses in the study were performed using SPSS 18.0 software (SPSS Inc., Chicago, IL, USA). In all of the analyses, the differences were considered statistically significant when p < 0.05 and extremely significant when p < 0.01. The figures were modified and edited using Origin 2023 and Adobe Illustrator 2020 [29].

3. Results

3.1. Walnut Orchard Soil Properties

Table 2 shows that the soil of the four walnut orchards is alkaline, ranging from 7.36 to 7.52, with an average value of 7.50. The average contents of SOM and TN in the walnut orchards at different altitudes were 27.2 and 0.04 g kg−1, respectively, and the contents of SOM and TN at 3112 m were the highest. The average contents of soil available N, available P, available K, and slowly available K were 2.02, 7.74, 94.1, and 1256.6 mg kg−1, respectively, indicating that the supply capacity of available N and available P in the soil of the main walnut orchards on the Qinghai–Tibet Plateau was seriously low, while the supply capacity of soil K was higher compared to that of the other nutrients.

3.2. Alpha Diversity of Soil Bacteria and Fungi in Walnut Orchards

The rarefaction curve was used to determine the variation trend of the sample alpha diversity during data leveling analysis. As shown in Figure 1, the curves of the four groups of soil samples all tended to flatten after the depth reached 45,000, indicating that the alpha diversity was close to saturation. The size of the alpha diversity index is related to the depth of the ASV data flattening. As shown in Figure 1, the sequencing results adequately reflected the diversity contained in the current sample and a large number of undiscovered new species could not be detected as they continued to increase.
As shown in Table 3, the altitude significantly affected the richness of the bacterial community and the richness and diversity of the fungal community but had no significant effect on the diversity of the bacterial community. For the bacterial group, the highest Chao1 index and species index values were 4172 and 4151 at the altitude of 3364 m, respectively. For the fungal group, the highest Chao1 index and species index values were 884 and 884 at the altitude of 3248 m, respectively. There was no significant difference between the Simpson index and Shannon index values of the bacterial community at each altitude, while the Simpson index and the Shannon index of the fungal community reached their maximum values at 3248 m, which were 0.9854 and 7.41, respectively. A correlation analysis (Table S1) was conducted between the altitude and the Chao1, species, Simpson, and Shannon indices of the bacterial and fungal communities, and the correlation coefficients were 0.066, 0.066, −0.212, and −0.374 for the bacterial community, respectively, and −0.409 (p < 0.05), −0.406 (p < 0.05), −0.298, and −0.225 for the fungal community, respectively. The results showed that there was a significant negative correlation between the richness of fungal community and the altitude. The diversity of the bacterial community and the fungal community decreased with the increase in altitude, but the difference was not significant.

3.3. Composition of Soil Bacteria and Fungi in Walnut Orchards

As shown in Figure 2a, from the bacterial phylum level, Actinobacteria, Acidobacteria, Proteobacteria, and Chloroflexi had the highest relative abundance in the four walnut orchards, belonging to the predominant bacterial community (relative abundance > 10.0%) with averages of 33.83%, 17.20%, 15.37%, and 14.47%, respectively. On the other hand, the relative abundances of Planctomycetes, Gemmatimonadetes, Thaumarchaeota, Verrucomicrobia, Rokubacteria, Bacteroidetes, and Firmicutes were lower, with averages of 4.79%, 4.09%, 2.38%, 1.78%, 1.15%, 1.01%, and 1.00%, respectively. With the increase in altitude, the relative abundance of Actinobacteria increased gradually, while that of Acidobacteria and Proteobacteria decreased gradually. For Chloroflexi, the variation in altitude was not obvious in the four treatments. Ascomycota was the dominant species, with an average relative abundance of 74.1%, followed by Basidiomycota and Mortierellomycota, with an average relative abundance of 8.7% and 5.9%, respectively (Figure 2b). The relative abundances of the other phyla were lower than 0.5%. With the increase in altitude, the relative abundance of Ascomycota increased significantly, while that of Basidiomycota, Mortierellomycota, Glomeromycota, and Chytridiomycota showed a decreasing tendency.
At the level of bacterial phylum (Figure 3a), Proteobacteria, Planctomycetes, Latescibacteria, Bacteroidetes, Elusimicrobia, Nitrospirae, and Patescibacteria were significantly altered taxa in the 3112 m treatment. Acidobacteria, Verrucomicrobia, and Rokubacteria were found to be sensitive to the 3248 m treatment. Intriguingly, the predominant Gemmatimonadetes, Firmicutes, Thaumarchaeota, Euryarchaeota, and Entotheonellaeota were significantly altered in the 3364 m treatment and only Actinobacteria, Chloroflexi, Armatimonadetes, and Cyanobacteria were positively altered in the 3550 m treatment. At the level of fungal phylum (Figure 3b), Rozellomycota, Chytridiomycota, and Mucoromycota were significantly altered taxa in the 3112 m treatment. Mortierellomycota, Glomeromycota, and Kickxellomycota were sensitive to the 3248 m treatment. Conversely, only Ascomycota was affected by the 3364 m treatment. Basidiomycota and Calcarisporiellomycota were clearly altered in the 3550 m treatment.
The species composition of bacteria and fungi at the genus level are shown in Figure 4. The results indicated that there were differences between the treatments to various degrees. At the bacterial genus level, the dominant groups were Subgroup_6, KD4-96, 67-14, g__RB41, Gaiella, JG30-KF-CM45, and Solirubrobacter, with average relative abundances of 8.98%, 5.46%, 3.79%, 3.50%, 2.77%, 2.61%, and 2.01%, respectively, followed by MB-A2-108, Rubrobacter, Nitrososphaeraceae, Blastococcus, Conexibacter, Rokubacteriales, TK10, Pseudonocardia, Nocardioides, and MND1, with average relative abundances between 1% and 2% (Figure 4a). At the fungal genus level, the dominant species were Penicillium, Mortierella, Fusarium, Humicola, Didymella, and Gibberella, having average relative abundances of 8.67%, 5.95%, 3.78%, 2.90%, 2.43%, and 2.13%, respectively, followed by Staphylotrichum, Cryptocoryneum, Tausonia, Solicoccozyma, Purpureocillium, and Hygrocybe, with an average relative abundance of 1.39%.

3.4. Beta Diversity of Soil Bacteria and Fungi in Walnut Orchard Soil

A principal coordinate analysis (PCA) can reflect the diversity difference of the soil microbial community on a two-dimensional coordinate diagram, and the contribution rate of differences can be explained by the horizontal axis 1 and the vertical axis 2. For the bacterial community, the first principal component (PCA1) and the second principal component (PCA2) accounted for 46.1% and 30.4% of the differences, respectively, for a total of 76.5%. The distance between the treatments was relatively long, indicating that the altitude caused great changes in the bacterial community composition. For the fungal community (Figure 5b), the first and second principal components explained 40.4% and 31.4% of the variance, respectively, with a combined contribution of 71.5%. As can be seen from Figure 2b, the distance between the fungal communities at 3112 m and 3248 m above sea level is small, indicating a small difference between them. However, the distance between these fungal communities and the fungal communities at 3364 m and 3550 m is longer and the community composition is significantly different.
In the Venn diagram (Figure 6a), there are a total of 15,155 bacterial ASVs in the soil samples of four groups in the Tibetan Plateau walnut orchards, among which 3720, 3553, 4147, and 3735 ASVs are contained at 3112 m, 3248 m, 3364 m, and 3550 m, respectively. There were 2636 unique ASVs in the walnut orchard soil at 3112 m, accounting for 21.7% of the total ASVs. The proportions of unique ASVs at the 3248 m, 3364 m, and 3550 m sites were 2215 (18.2%), 2762 (22.7%), and 2543 (20.9%), respectively. The number of ASVs that are common to all four plots was 248 (2.0%). Similarly, in the fungal Venn diagram (Figure 6b), a total of 2769 ASVs were found in the soil samples of four groups in the Tibetan Plateau walnut orchards, among which 725, 875, 581, and 588 ASVs were found at 3112 m, 3248 m, 3364 m, and 3550 m, respectively. There were 507 ASVs in the walnut orchard soil at 3112 m, accounting for 20.6%. The proportion of ASVs in the 3248 m, 3364 m, and 3550 m samples were 604 (26.9%), 373 (16.6%), and 402 (17.9%), respectively. The total number of ASVs common to all four sites was 37 (1.6%).
A heatmap analysis can be used to cluster ASVs with different relative abundances at the level of genus classification, and the diversity of microbial species can be indicated by different colors. As shown in Figure 7, the altitude significantly affected the distribution and relative abundances of the dominant genera of bacteria and fungi in the soil of the four walnut orchards, and the species of bacteria and fungi in walnut orchards with adjacent altitudes were more similar. The number of dominant bacterial genera at 3112 m, 3248 m, 3364 m, and 3550 m reached 21, 12, 7, and 5, respectively, while the number of dominant fungi reached 20, 9, 9, and 8, respectively. It was found that with the increase in altitude, the number of dominant genera of soil bacteria and fungi gradually decreased and there were significant differences in the distribution.

3.5. Correlation of Dominant Microbial Communities with Altitude

As shown in Table 4, Actinobacteria in the bacterial community was significantly positively correlated with altitude (p < 0.01); Proteobacteria and Planctomycetes were significantly negatively correlated with altitude (p < 0.01); and Acidobacteria, Verrucomicrobia, and Bacteroidetes were significantly negatively correlated with altitude (p < 0.05). For the fungal group, Olpidiomycota and Zoopagomycota were significantly positively correlated with altitude (p < 0.05) and Chytridiomycota and Rozellomycota were significantly negatively correlated with altitude (p < 0.05). There was no significant correlation between the altitude and the other bacterial and fungal phyla.

3.6. Correlation between Dominant Microbial Communities and Soil Properties

The redundancy analysis (RDA) (Pseudo-F = 412, p = 0.002 **) showed that PCA 1 and PCA 2 explained 89.1% and 6.4% of the total variance in the soil bacterial community composition, respectively. The phyla Rokubacteria, Acidobacteria, Verrucomicrobia, Planctomycetes, and Proteobacteria were clustered together to the edge of soil available N, TN, available K, and SOM. By contrast, the phyla Gemmatimonadetes, Actinobacteria, and Thaumarchaeota were highly correlated with slowly available K and pH. The available N, SOM, TN, and available K had a noteworthy impact on the bacterial community and explained the variation by 44.3%, 32.2%, 6.4%, and 6.4%, respectively (Figure 8a). RDA (Pseudo-F = 536, p = 0.002 **) showed that PCA 1 and PCA 2 explained 92.1% and 5.8% of the total variance in the soil fungal community composition, respectively (Figure 8b). Mortierellomycota, Glomeromycota, Rozellomycota, Chytridiomycota, and Mucoromycota had a positive correlation with soil TN, SOM, available N, available P, and available K; Ascomycota was highly correlated with slowly available K and pH. Available N, SOM, TN, available P, and available K explained the variation by 44.1%, 15.3%, 9.3%, 8.1%, and 6.5%, respectively.

4. Discussion

The plateau walnut is the main perennial economic forest plant cultivated in southeastern Tibet, and its growth and reproduction processes are closely related to the natural environment and soil microorganisms [1,24,27]. As the disintegrator in soil, microorganisms participate in the mineralization process and in the biological fixation of nutrients, thereby affecting the absorption and utilization of nutrient elements via walnut roots [18]. The soil microbial diversity and composition can also reflect the stability of the soil community structure, as well as the soil health and fertility. Changes in altitude cause changes in the microclimate, vegetation type, soil parent material, and soil type (Table 1 and Table 2), resulting in differences in the soil nutrient content and the microbial community structure and diversity [30,31]. In this study, high-throughput sequencing was used to analyze the soil bacterial and fungal communities of walnut orchards at different altitudes in Gyaca county (Table 3). The results showed that, with the exception of the bacterial richness index, the diversity of the bacterial community and the richness and diversity indices of the fungal community all showed a downward trend with the increase in altitude, but the difference was not significant. He et al. [14] showed that the richness of the soil bacterial community on the vertical gradient of the Daiyun Mountain was greatly affected by the altitude gradient. As the altitude is highly correlated with the annual mean temperature and the annual precipitation, this indicates that the soil bacterial community is more affected by climate than by geochemical properties or soil structure. Wang et al. [31] also found that with the increase in altitude, the Shannon index and Chao1 index of the bacterial and fungal communities decreased significantly, which was consistent with the findings of the present study which indicated that the increase in altitude reduced the diversity of the microbial community among the Tibetan Plateau walnut orchards in Tibet.
As shown in Figure 6, the soil microorganisms in the walnut orchards at different altitudes were affected by environmental changes and their community composition gradually varied, with only 1.6% of species found in all four sites. However, the dominant bacteria in the soil bacterial community in each treatment were Actinobacteria, Acidobacteria, Proteobacteria, and Chloroflexi (Figure 2), which were similar to the findings of Yang et al. [13]. That is, the dominant bacteria species were not affected by the altitude. Correlation analysis showed that the relative abundances of Actinomyces and Chloromyces were positively correlated with altitude, while the relative abundances of Acidobacteria and Proteobacteria were negatively correlated with altitude (Table 4). Proteobacteria belong to eutrophic bacteria, which grow in environments with good nutritional conditions and can indicate soil fertility characteristics [32]. In addition, some Proteobacteria perform biological nitrogen fixation and can promote soil nitrogen cycling. Acidobacteria belong to oligotrophic bacteria, which can grow in nutrient-deficient soil environments and have the ability to degrade inactive organic carbon sources. Actinomycetes are mostly saprophytic bacteria, which can participate in soil nutrient cycling and energy conversion and are the main biological population involved in litter degradation [33]. The SOM content was high in the highland walnut orchards, while the available nutrient contents of mineral elements such as nitrogen, phosphorus, and potassium were relatively low (Table 2). Therefore, the plateau fertility depends more on the mineralization of deciduous organic matter and the return of available nutrients (e.g., fallen leaves and residual roots). In the bacterial community, the relative abundances of dominant bacteria were markedly different according to increases in altitude. Especially, the Vicinamibacterales and Burkholderiales (Figure 3) have a strong soil nitrogen and phosphorus activation abilities [34].
Fungal communities and their activity are closely related to soil litter species and decomposition rates [15]. Ascomycota was the dominant fungal group in the soil fungal community of the Tibetan Plateau walnut orchards, followed by Basidiomycota and Mortierellomycota (Figure 2b). The relative abundances of Ascomycetes and Basidiomycetes increased with the increase in altitude but not significantly. Ascomycota and Sporospora are eutrophic fungi with a strong adaptability to the environment that can rapidly metabolize organic substrates [16]. Basidiomycetes belong to the oligotrophic fungi and are rich in ligninolytic and cellulose enzymes. Their mycelium has a widespread distance and their relative abundance increases with the increase in altitude [24,25,26,27]. Therefore, fungi and their mycelium play an important role in assisting nutrient absorption at the highest altitude (3550 m) walnut orchard.
In the mountains, the soil microbial community structure is mainly regulated by temperature and rainfall, followed by the physical and chemical properties of soil [14,15,30]. From the results of Figure 8, the limitations of soil N and P have significant effects on the bacterial and fungal communities; hence, additional nutritional supplements are necessary for walnut tree growth [22]. However, different from other walnut-producing areas, the plateau walnut is mainly produced organically without any chemical fertilizer. Therefore, the application of bio-organic fertilizer that is rich in nitrogen and phosphorus can not only increase walnut yield and improve soil fertility, but also maintain the microbial diversity in the Tibet Autonomous Region.

5. Conclusions

The soil microbial communities in natural plateau walnut orchards varied along the elevation gradient. Actinobacteria, Acidobacteria, Proteobacteria, and Chloroflexi had the highest relative abundances in the four walnut orchards, belonging to the predominant bacterial community. With the increase in altitude, the relative abundance of Actinobacteria increased gradually, while that of Acidobacteria and Proteobacteria decreased gradually. The dominant fungal phyla were Ascomycota, Basidiomycota, and Mortierellomycota. With the increase in altitude, the relative abundance of Ascomycota increased significantly. Actinobacteria in the bacterial community, and Olpidiomycota and Zoopagomycota in the fungal community, were significantly positively correlated with altitude. Most dominant bacterial and fungal phyla were significantly negatively correlated with altitude. The soil nitrogen and phosphorus availabilities are the main limiting factors of the soil microbial community diversity at higher altitudes in the natural walnut orchards of southeast Tibet.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/land12071419/s1, Table S1: Correlation coefficient between altitude and microbial alpha diversity.

Author Contributions

Writing—original draft preparation, R.Y.; writing—review and editing, Y.G. and J.L.; formal analysis, P.L.; investigation, X.Z., Z.S. and R.Y. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported in part by the Open Fund of Key Laboratory of Crop Water Requirement and Regulation, Ministry of Agriculture and Rural Affairs, China (ZWS2023-02).

Data Availability Statement

Not applicable.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Rarefaction curves of bacteria (a) and fungi (b) in walnut orchards at different altitudes.
Figure 1. Rarefaction curves of bacteria (a) and fungi (b) in walnut orchards at different altitudes.
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Figure 2. Relative abundance of major taxonomic groups for (a) bacteria and (b) fungi at the phylum level in walnut orchard soil.
Figure 2. Relative abundance of major taxonomic groups for (a) bacteria and (b) fungi at the phylum level in walnut orchard soil.
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Figure 3. Significant changes in bacterial (a) and fungal (b) key phylotypes identified using Linear discrimination analysis Effect Size (LEfSe) in walnut orchard soil.
Figure 3. Significant changes in bacterial (a) and fungal (b) key phylotypes identified using Linear discrimination analysis Effect Size (LEfSe) in walnut orchard soil.
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Figure 4. Relative abundances of major taxonomic groups for (a) bacteria and (b) fungi at the genus level in walnut orchard soil.
Figure 4. Relative abundances of major taxonomic groups for (a) bacteria and (b) fungi at the genus level in walnut orchard soil.
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Figure 5. Principal coordinate analysis (PCA) plot of the dissimilarity between the walnut orchards for (a) bacteria and (b) fungi based on Bray–Curtis differences. Different colors and geometric shapes identify the treatments and the sample points.
Figure 5. Principal coordinate analysis (PCA) plot of the dissimilarity between the walnut orchards for (a) bacteria and (b) fungi based on Bray–Curtis differences. Different colors and geometric shapes identify the treatments and the sample points.
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Figure 6. Venn diagram of (a) bacterial and (b) fungal operational taxonomic units from walnut orchard soil samples.
Figure 6. Venn diagram of (a) bacterial and (b) fungal operational taxonomic units from walnut orchard soil samples.
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Figure 7. Genus-level variations in dominant (a) bacteria and (b) fungi among walnut orchards at four different elevations.
Figure 7. Genus-level variations in dominant (a) bacteria and (b) fungi among walnut orchards at four different elevations.
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Figure 8. Redundancy analysis (RDA) of soil properties and main (a) bacterial and (b) fungal communities at the phylum level in soils. Red lines represent soil properties and blue lines represent the bacterial and fungal phylum-level taxonomy.
Figure 8. Redundancy analysis (RDA) of soil properties and main (a) bacterial and (b) fungal communities at the phylum level in soils. Red lines represent soil properties and blue lines represent the bacterial and fungal phylum-level taxonomy.
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Table
Table 1. Basic information of walnut orchards at different altitudes.
Table 1. Basic information of walnut orchards at different altitudes.
Walnut OrchardAltitudeVegetation TypeParent MaterialAgrotype
Ciren village, Anrao town3112 mWalnut, rapeseed, highland barleysedimentCalcareous alluvial soil
Longba village, Gyaca town3248 mWalnut, grassland, shrubgluteniteBrush prairie soil
Lagang village, Anrao town3364 mWalnut, grasslanddeluviumPlateau prairie soil
Sangzhu village, Anrao town3550 mWalnut, grasslandeluviumMountain meadow soil
Table
Table 2. Soil properties of four walnut orchards at different altitudes.
Table 2. Soil properties of four walnut orchards at different altitudes.
AltitudepHSOM
(g kg−1)		TN
(g kg−1)		Available N
(mg kg−1)		Available P
(mg kg−1)		Available K
(mg kg−1)		Slowly Available K (mg kg−1)
3112 m7.42 ab38.9 a0.06 a3.99 a21.73 a194.8 a1126.2 b
3246 m7.36 b32.8 a0.05 a2.13 b1.91 d50.8 c780.9 c
3364 m7.51 a16.5 c0.02 c0.86 d3.31 c57.4 c2539.7 a
3550 m7.52 a20.7 b0.04 b1.12 c4.03 b73.5 b579.6 d
Note: Different letters for the same item indicate p < 0.05 (significant differences).
Table
Table 3. Alpha diversity indices of bacteria and fungi in walnut orchards at different altitudes.
Table 3. Alpha diversity indices of bacteria and fungi in walnut orchards at different altitudes.
Microbe TypeAltitudesRichness IndexDiversity IndexCoverage
Chao1SpeciesSimpsonShannon
Bacteria3112 m3750 b3725 b0.9986 a10.79 a0.999 a
3248 m3561 c3556 c0.9987 a10.76 a1.000 a
3364 m4172 a4151 a0.9987 a10.85 a0.999 a
3550 m3769 b3746 b0.9986 a10.65 a0.999 a
Fungi3112 m731 b730 b0.9733 a6.54 b1.000 a
3248 m884 a884 a0.9854 a7.41 a1.000 a
3364 m591 c589 c0.8732 b5.54 c1.000 a
3550 m589 c589 c0.9301 a6.10 b1.000 a
Note: Different letters for the same item indicate p < 0.05 (significant differences).
Table
Table 4. Pearson correlation coefficient between altitude and soil bacterial and fungal phyla.
Table 4. Pearson correlation coefficient between altitude and soil bacterial and fungal phyla.
BacteriophytaCorrelationEumycophytaCorrelation
Actinobacteria0.819 **Ascomycota0.069
Acidobacteria−0.673 *Basidiomycota0.070
Proteobacteria−0.809 **Mortierellomycota−0.285
Chloroflexi0.193Glomeromycota−0.499
Planctomycetes−0.837 **Chytridiomycota−0.655 *
Gemmatimonadetes0.462Olpidiomycota0.672 *
Thaumarchaeota−0.015Zoopagomycota0.685 *
Verrucomicrobia−0.527 *Kickxellomycota−0.014
Rokubacteria−0.396Mucoromycota−0.089
Bacteroidetes−0.605 *Rozellomycota−0.645 *
Note: * and ** indicate significant differences at the p < 0.05 and p < 0.01 levels, respectively.
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Yan, R.; Zhao, X.; Li, P.; Si, Z.; Gao, Y.; Li, J. Composition and Diversity of Soil Microbial Communities in Walnut Orchards at Different Altitudes in Southeastern Tibet. Land 2023, 12, 1419. https://doi.org/10.3390/land12071419

AMA Style

Yan R, Zhao X, Li P, Si Z, Gao Y, Li J. Composition and Diversity of Soil Microbial Communities in Walnut Orchards at Different Altitudes in Southeastern Tibet. Land. 2023; 12(7):1419. https://doi.org/10.3390/land12071419

Chicago/Turabian Style

Yan, Ruyu, Ximei Zhao, Penghui Li, Zhuanyun Si, Yang Gao, and Jifu Li. 2023. "Composition and Diversity of Soil Microbial Communities in Walnut Orchards at Different Altitudes in Southeastern Tibet" Land 12, no. 7: 1419. https://doi.org/10.3390/land12071419

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