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Article

A Treasure Trove of Urban Microbial Diversity: Community and Diversity Characteristics of Urban Ancient Ginkgo biloba Rhizosphere Microorganisms in Shanghai

by
Jieying Mao
1,2,3,
Qiong Wang
1,2,
Yaying Yang
1,2,4,
Feng Pan
5,
Ziwei Zou
1,2,
Xiaona Su
1,2,
Yi Wang
1,2,
Wei Liu
1,2,* and
Yaohua Tang
6,*
1
Jiangxi Provincial Key Laboratory of Conservation Biology, Nanchang 330045, China
2
School of Art and Landscape, College of Forestry, Jiangxi Agricultural University, Nanchang 330045, China
3
Ningbo Yongneng Biomass Energy Development Co., Ltd., Ningbo 315000, China
4
Ganzhou Vegetable and Flower Rrerarch Institute, Ganzhou 341413, China
5
Jiangxi Academy of Water Science and Engineering, Nanchang 330029, China
6
Shanghai Municipal Landscape Management and Guidance Station, Shanghai 200020, China
*
Authors to whom correspondence should be addressed.
J. Fungi 2024, 10(10), 720; https://doi.org/10.3390/jof10100720
Submission received: 9 July 2024 / Revised: 26 September 2024 / Accepted: 10 October 2024 / Published: 16 October 2024
(This article belongs to the Special Issue Fungal Communities in Various Environments)
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Abstract

Rapid urbanization has exerted immense pressure on urban environments, severely constraining the growth of ancient trees. The growth of ancient trees is closely linked to the microbial communities in their rhizospheres, and studying their community characteristics may provide new insights into promoting the growth and rejuvenation of ancient trees. In this study, the rhizosphere soil and root systems of ancient Ginkgo biloba trees (approximately 200 years old) and adult G. biloba trees (approximately 50 years old) in Shanghai were selected as research subjects. Phospholipid fatty acid (PLFA) analysis and high-throughput sequencing were employed to investigate the diversity of microbial communities in the G. biloba rhizosphere. The results indicated that the 19 PLFA species selected to characterize the soil microbial community structure and biomass were present in the rhizosphere soil of both ancient and adult G. biloba trees. However, the total microbial biomass and the microbial biomass in the rhizosphere soil of ancient G. biloba were lower than the microbial biomass in the rhizosphere soil of adult G. biloba. The biomasses of Gram-negative bacteria (G−), arbuscular mycorrhizal fungi (AMF), and protozoans (P) were significantly different. Total phosphorus, organic matter, and pH may be the key factors influencing the soil microbial community in the rhizosphere zone of ancient G. biloba. An in-depth study of AMF showed that the roots and rhizosphere soil of G. biloba contained abundant AMF resources, which were assigned to 224 virtual taxa using the MaarjAM reference database, belonging to four orders, ten families, and nineteen genera. The first and second most dominant genera were Glomus and Paraglomus, respectively. Archaeospora and Ambispora were more dominant in the rhizosphere than the roots. Furthermore, the abundance of live AMF was significantly higher in ancient G. biloba than in adult G. biloba. Therefore, future research should focus on the improvement of soil environmental characteristics and the identification and cultivation of indigenous dominant AMF in the rhizosphere of ancient G. biloba, aiming for their effective application in the rejuvenation of ancient trees.

1. Introduction

According to China’s National Technical Regulations for the Census of Ancient and Famous Trees, published in 2001, ancient trees are defined as trees that are 100 years old and older [1]. These trees carry important historical memories and research value, making them a valuable natural and cultural heritage resource [2]. However, the vigor of ancient trees gradually weakens after a long period of growth. Natural disasters, pests, and diseases are likely to cause harm to ancient trees. Moreover, old trees that grow in cities have an even less optimistic outlook. Ginkgo biloba is an endemic tree species in China that only blossoms and bears fruit after about 20 years [3]. The development of flowers occurs when G. biloba reaches adulthood [4]. With a large population, G. biloba is an important ancient and famous tree resource in China. G. biloba is the most abundant ancient tree species in Shanghai [5]. However, according to the Shanghai Municipal Administration of Greening and Amenities, approximately one-third of ancient G. biloba in Shanghai exhibit signs of poor growth owing to natural and anthropogenic influences.
Soil quality is among the most important factors affecting the growth of ancient trees. Problems such as soil compaction, heavy metal pollution, mineral nutrient scarcity, and imbalances in rationing affect the health of ancient trees, resulting in the slow growth of their root systems and an insufficient supply of nutrients. The most active components of the soil are microorganisms, which can interact with the soil. The soil microbiota is also an important indicator of soil quality [6]. Therefore, there is a strong link between the soil microbiota and the growth of ancient trees. However, studies have shown that there are significant differences in the structure of the microbiota between the rhizosphere and non-rhizosphere soils of G. biloba, and the profile of soil fungi is complex and inconsistent between these soils [7].
Arbuscular mycorrhizal fungi (AMF) are a group of endophytic fungi widely distributed in terrestrial ecosystems [8]. AMF are present in the soil as spores and mycelia. AMF account for the largest proportion of the fungal biomass in soil and directly affect the microbial community structure through mycelial secretions that cause changes in plant root secretions [9]. Previous studies have confirmed that AMF infesting the root system of G. biloba is a common phenomenon that forms a mutually beneficial symbiotic system with the host. Fontana [10] found that G. biloba on the campus of the Università degli Studi di Torino, Italy, exhibited numerous intracellular mycelia. Wu and Wei [11] found that the development of clumped mycorrhizae in grafted G. biloba seedlings was significantly better than that seen in live seedlings grown through seed germination. Yuan et al. [12] determined that the AMF of G. biloba growing on artificial coasts exhibited a P-type structure. In a symbiotic system formed with the host plant, AMF can absorb mineral nutrients through mycelial extensions into soil crevices that are inaccessible to the plant root system for transfer to the host plant [13,14]. Additionally, AMF have various beneficial effects on soil, including improving the physical structure, stabilizing organic carbon, activating nutrients, and maintaining pore space [15,16].
Tong et al. [17] applied exogenous mycorrhizal fungi to ancient G. biloba in Shanghai and found that AMF inoculation significantly promoted the growth of ancient trees, with enhanced soil fertility detected four years later. This suggests that mycorrhizal fungi can promote the rejuvenation of ancient G. biloba. In fact, indigenous mycorrhizal fungi in the rhizosphere soil of plants may be more adaptable to the soil environment and function more efficiently than exogenous mycorrhizal fungi because they have a closer relationship with the host plants [18]. Therefore, exploring the diversity of microbial communities, especially AMF communities, may provide a new direction for promoting the growth of ancient G. biloba and for salvage and rejuvenation.
After hundreds or even thousands of years of growth, ancient G. biloba may have a more stable microbial community in its rhizosphere than younger adult G. biloba. In this study, it was hypothesized that (1) ancient G. biloba trees accumulate more microbial species and numbers in the rhizosphere soil than younger adult G. biloba; and (2) AMF are present in both the roots and rhizosphere soils of G. biloba but may differ considerably in terms of species and number between ancient and younger adult G. biloba.
To test these hypotheses, phospholipid fatty acid (PLFA) technology was employed to detect microbial taxa in the rhizosphere soil of ancient and younger adult G. biloba in Shanghai. High-throughput sequencing technology was utilized to reveal the community composition and diversity of AMF in the rhizosphere of G. biloba in depth. It is hoped that this study will provide a research basis for identifying microbial taxa in the rhizosphere soils of ancient G. biloba, as well as for the preparation of mycorrhizal agents to promote the growth and rejuvenation of ancient trees.

2. Materials and Methods

2.1. Site and Experimental Design

The sampling sites were located in the Jiading District and Qingpu District (31°9′14′′ N–31°23′3′′ N, 121°8′35′′ E–121°27′5′′ E) of Shanghai, China (Figure 1). The region has a northern subtropical monsoon climate with warm weather and ample precipitation. The average annual temperature is 17.7 °C, and precipitation is concentrated from April through October, with an average annual precipitation of 1726 mm.
To explore the symbiosis between AMF and G. biloba, two types of G. biloba were selected for the study: eight ancient G. biloba approximately 200 years old, and five adult G. biloba approximately 50 years old. The ages of ancient G. biloba trees were determined according to the times recorded on the protection plates for ancient and famous trees established by the Shanghai Municipal Administration of Greening and Amenities, which were obtained through a literature review [19]. The ages of adult G. biloba trees were estimated based on the landscaping projects implemented by the Shanghai Municipal Administration of Greening and Amenities at that time. None of these trees were treated with mycorrhizal fungicides. Fieldwork was performed on 17 May 2021 and 15 September 2022, and the relevant information about each tree was recorded (Table 1). The drip lines of G. biloba were located in four directions: east, west, south, and north. During sample collection, 150 g of mixed soil and roots were collected to a depth of approximately 30 cm. Samples from all four directions were placed on a newspaper sheet to remove plant debris and stones. The samples were then mixed thoroughly in a sealing bag to form a composite sample, and this process was repeated three times for each tree. To prevent contamination, the shovel was cleaned to remove soil particles and disinfected with 70% ethanol before and after digging, and sterile gloves were utilized during the removal of plant debris and stones.
For sample preservation, it was necessary to separate the roots from the soil in each sample and rinse the root surface. The samples were labeled as the roots of ancient G. biloba (GYXG), the rhizosphere soil of ancient G. biloba (GYXT), the roots of adult G. biloba (XYXG), and the rhizosphere soil of adult G. biloba (XYXT). Each soil sample was equally divided into three parts, one of which was given to the South China Botanical Garden for the PLFA assay based on the methodology referenced from Bossio et al. [20] and analyzed using the MIDI Sherlock® Microbial Identification System 6.0. Another part, together with the roots of G. biloba, was sent to Novogene for high-throughput sequencing. As the samples were not immediately available for PLFA and high-throughput sequencing assays, they were stored temporarily at −20 °C. The final part was used to determine the chemical properties, stored at room temperature (20–25 °C), sieved (2 mm), spread flat in the laboratory, and allowed to dry naturally.

2.2. Experimental Methods

2.2.1. Soil Chemical Analyses

The soil was digested using the HClO4-H2SO4 decoction method before the analysis of the total nitrogen (TN), total phosphorus (TP), and total potassium (TK) contents. The TN was analyzed using a flow analyzer (AutoAnalyzer III, Bran + Luebbe GmbH, Hamburg, Germany). The TP was measured using the molybdenum antimony anti-colorimetric method [21], and the TK was determined using a flame photometer (FP640, Jingke, Shanghai, China). The potassium dichromate-potassium external heating method was employed to determine the organic matter (OM) content. The soil pH was measured using a pH meter (PB-10, Sartorius AG, Göttingen, Germany), and the soil conductivity was measured using a conductivity meter (DDS-307, Yidian Corporation, Shanghai, China). Each soil sample was replicated three times with a blank control.

2.2.2. PLFA Analysis

The quantified internal standard, normal nonadecanoic acid (19:0), was employed to dissolve the extracted and processed fatty acid methyl esters obtained from the soil samples. Gas chromatography (Agilent 7890A, Santa Clara, CA, USA) was utilized to obtain the response values for the characteristic peaks of each fatty acid region [22]. A reference to previously compiled PLFA markers [23] was used to select 19 specific types (Table 2) to characterize the structure and biomass of the soil microbial community. Additionally, the total microbial biomass (T-PLFA) for the six microbial groups was calculated, and ratios including the ratio of Gram-positive bacteria to Gram-negative bacteria (G+/G−), the environmental pressure (cy17:0c/16:1ω7c), and the ratio of fungi to bacteria (F/B) were determined.

2.2.3. High-Throughput Sequencing Methods for AMF

Five grams each of soil and root samples were used for DNA extraction. A total of 1000 μL of cetyltrimethylammonium bromide (CTAB) lysate, 20 μL of lysozyme, and 1 μL of RNase A were used to extract the DNA of AMF from roots and soil according to the CTAB method [24]. There were three biological replicates for each sample. The quality of DNA extraction was analyzed using 1.2% agarose gel electrophoresis [25,26]. DNA was amplified using polymerase chain reaction (PCR) with a target fragment length of 280 bp. The AMF-specific primers AMV4.5NF (5′-AAG CTC GTA GTT GAA TTT CG-3′) and AMDGR (5′-CCC AAC TAT CCC TAT TAA TCA T-3′) were utilized to accomplish this process [27,28,29]. Gel electrophoresis was performed on the amplification products to obtain valid samples for the amplification of specific fragments. The assay conditions were a 2% gel concentration, 80 V, and 40 min of electrophoresis time.
Paired-end sequencing was performed on the samples using a sequencer (Illumina NovaSeq 6000, San Diego, CA, USA). Due to the possibility of sequencing misalignment in the raw data, splicing and filtering were performed to obtain valid data. DADA2 was employed for denoising, and sequences with an abundance of less than five were filtered out. Amplicon sequence variant (ASV) feature sequences with nearly 100% similarity were obtained through clustering [30]. Each ASV feature sequence was aligned with the Nt database to eliminate non-Glomeromycota sequences. This was followed by a BLAST comparison of the remaining representative sequences on MaarjAM (https://maarjam.botany.ut.ee/, accessed on 10 March 2023) to attribute the sequences to the AMF virtual taxa and determine the genus information [31,32]. This classification was a virtual classification based on small subunit rRNA genes in the MaarjAM database [33]. A virtual taxon was the classification unit, representing a well-supported monophyletic clade with sequence similarity within the clade exceeding a threshold of 97% [34]. Analyses including the ASV abundance, Venn diagrams, alpha diversity, and beta diversity were performed to obtain information on the absolute and relative abundances, shared and unique ASVs, and diversity indices, including the species richness (Chao1), Shannon diversity (Shannon), Simpson’s diversity (Simpson), and evenness (Pielou_e) indices [35].

2.3. Data Analysis

Zone mapping was performed using ArcGIS 10.8 (Environmental Systems Research Institute, Redlands, CA, USA). An independent samples t-test was utilized to analyze the variability of the soil microbial biomass and soil microbial community structure ratio. One-way analysis of variance (ANOVA) and Duncan’s multiple comparisons were used to complete the analysis of the AMF community data within the roots and soil of G. biloba, including the soil chemical, absolute, and relative abundances, shared and unique ASVs, and diversity indices such as Chao1, Shannon, Simpson, and Pielou_e. Differences were considered significant at p < 0.05 and highly significant at p < 0.01. Pearson’s coefficients were used to analyze the soil microbial biomass and correlation factors representing habitat conditions, as well as between AMF species and correlation factors representing habitat conditions. All statistical analyses were performed using SPSS version 22 (IBMSPSS Statistics, Armonk, NY, USA). These statistics were used to complete a drawing in Origin Pro 2022 (OriginLab, Hampden, MA, USA). Principal coordinate analysis (PCoA) was performed using the online platform Novogene Cloud (https://magic.novogene.com/ accessed on 27 July 2024) [36].

3. Results

3.1. Soil Environmental Traits

As displayed in Table 3, the results of soil chemical analyses indicated that there were significant differences between GYXT and XYXT. Except for the TN and OM, all other indices exhibited significant differences (p < 0.05). GYXT had significantly higher TN and TK contents than XYXT (p < 0.05), where the difference in TK was highly significant (p < 0.01). However, the TP, OM, pH, and electrical conductivity (EC) were lower in GYXT than in XYXT, with significant differences in the TP and EC (p < 0.05) and highly significant differences in pH (p < 0.01).

3.2. Microbial Biomass of the Rhizosphere Soil of G. biloba

In total, 19 specific PLFAs were detected in the 13 soil samples examined, and the same microbial species were present in XYXT and GYXT; however, there were large differences in their contents (Figure 2). XYXT had a higher biomass of F, G+, G−, A, AMF, and P than GXYT, and all except for G− were significantly different (p < 0.05). Among them, the biomass of G+, AMF, and P showed highly significant differences (p < 0.01). Calculating the total microbial biomass revealed that the total microbial biomass of GYXT was 82.61 nmol·g−1, which was highly significant (p < 0.01) and lower than that of XYXT. The highest content of PLFA biomarkers in XYXT and GYXT was found in G+, which accounted for 36.40% and 33.16% of the total PLFA biomarkers in the soils, respectively, and had the greatest predominance in the soils. The content of AMF in XYXT accounted for 5.14% of T-PLFA, which was slightly higher than that detected in GYXT (4.54%).

3.3. Relationship Between Soil Environmental Traits and Microbial Biomass

Pearson’s correlation analysis showed a significant correlation (p < 0.05) between the soil environmental traits and the microbial community structure. However, large differences in the soil environmental factors had significant effects on the microbial community structure in both ancient and adult G. biloba soils (Figure 3). In XYXT, the TK was significantly negatively correlated with AMF and P (p < 0.05), whereas the EC was significantly negatively correlated with F (p < 0.05) and significantly negatively correlated with G+ (p < 0.05). In GYXT, the TP and OM were significantly negatively correlated with F, G+, G−, AC, AMF, P, and the T-PLFA (p < 0.05). pH was significantly negatively correlated with P (p < 0.05) and highly negatively correlated with F, G+, G−, AC, AMF, and the T-PLFA (p < 0.01).
Fungi and bacteria are the most dominant microbial groups in soil, and the F/B and G+/G− ratios can indirectly respond to the survival environment of soil microorganisms and the diversity characteristics of bacterial communities. As shown in Table 4, the F/B ratio of GXYT was 0.009 higher than that of XYXT, and the G+/G− ratio was 0.246 lower than that of XYXT. The environmental pressure of GXYT was 0.360, which was significantly lower than that of XYXT (p < 0.05).

3.4. Analysis of the AMF Community in the Roots and Rhizosphere Soil of G. biloba

3.4.1. Composition of the AMF Community

Abundant AMF resources were found in the roots and rhizosphere soil of G. biloba (Table 5). In total, 224 AMF virtual taxa were obtained from all samples using high-throughput sequencing. These taxa belonged to four orders, ten families, and nineteen genera. Among them, the genus with the most identified species was Scutellospora, with five species, followed by Rhizophagus with four species, Paraglomus and Ambispora with three species, and Glomus, Septoglomus, Sclerocystis, Claroideoglomus, and Acaulospora with two species. Furthermore, Funneliformis, Dominikia, Kamienskia, Geosiphon, Diversispora, Redeckera, Gigaspora, Dentiscutata, and Pacispora all had only one species.
The most numerous virtual taxon was Glomus with 137 species, accounting for 72.49% of the total virtual taxa. There were 14 species of Paraglomus, 12 species of Claroideoglomus, 10 species of Archaeospora, 7 species of Acaulospora and Diversispora, and 1 each of Scutellospora, Ambispora, and Pacispora.

3.4.2. Abundance Analysis of AMF Community

The genus-level composition of the AMF community in the root and rhizosphere soils of G. biloba is shown in Figure 4. Glomus was the most dominant genus in all treatments, with the number of ASVs ranging from 225 to 267 across treatments and the relative species abundance ranging from 48.55% to 59.11%. The highest ASV numbers and relative species abundances were found in GYXG, and the ASV numbers and relative species abundances of Glomus in the roots were higher than those in the rhizosphere soil. The second most dominant genus in all treatments was Paraglomus, with ASV numbers ranging from 87 to 103 and a relative species abundance between 19.01% and 21.92%. In the rhizosphere soil, the third most dominant genus was Archaeospora, while in the roots, the third most dominant genus was Diversispora. Ambispora was the fourth most dominant genus in GYXT and XYXT, while the fourth most dominant genera in GYXG and XYXG were Archaeospora and Claroideoglomus, respectively. Additionally, Gigaspora, Acaulospora, Geosiphon, Scutellospora, Septoglomus, Dominikia, Sclerocystis, Rhizophagus, Funneliformis, Dentiscutata, Redeckera, Pacispora, and Kamienskia were present in small amounts in all treatments.

3.4.3. Venn Diagram Analysis of AMF Community

As shown in the Venn diagram, the numbers of total and unique ASVs in ancient G. biloba were greater than the corresponding ASV numbers detected in adult G. biloba (Figure 5). The highest numbers of total and unique ASVs in all treatments were found in GYXG, followed by GYXT and XYXT, and the lowest were found in XYXG. In addition, the number of shared ASVs in all treatments was low at only 244, accounting for 4.38% of the total number of ASVs. The highest number of shared ASVs between GYXT and XYXT was 725, and the lowest number of shared ASVs between XYXT and GYXG was 501.

3.4.4. Diversity of AMF Community

The diversity of the AMF community can be assessed using diversity indices such as Shannon, Simpson, Chao1, and Pielou_e (Figure 6). As shown in Figure 6, the median Chao1 index and median Shannon index of soil samples were higher than those of the root samples. The median Simpson index of adult G. biloba was higher than that of ancient G. biloba. The Pielou’s evenness index median was highest in XYXT (0.741) and lowest in XYXG (0.631), with a slight difference of 0.003 between the ancient G. biloba roots and the rhizosphere soil. However, there were no significant differences in the four diversity indices among treatments. Additionally, looking at the overall picture of the four indices, the dispersion of the indices in ancient G. biloba was greater than that in adult G. biloba. The skewness was stronger, and most were negatively skewed.

3.4.5. PCoA of the AMF Community

PCoA using the unweighted UniFrac distance matrix algorithm revealed that the AMF community structures in the root and rhizosphere soils of G. biloba were significantly different (Figure 7). The AMF community structures of GYXG and XYXG were more similar, and those of GYXT and XYXT were more similar. The PCoA results indicated that a small number of widely differing biological replicates appeared in GYXG and XYXG, but all were clustered in the first and second quadrants, were positively correlated with PC1, and were basically negatively correlated with PC2. The biological replicates of GYXT and XYXT were all well clustered together, clustered in the third and fourth quadrants, were negatively correlated with PC1, and were basically positively correlated with PC2.

3.4.6. Cluster Analysis of AMF Community

As shown in the cluster analysis heat map, the genera with relatively high abundances varied among all treatments (Figure 8). In the root treatments, seven genera had relatively high abundances in GYXG, namely Paraglomus, Claroideoglomus, Septoglomus, Sclerocystis, Diversispora, Gigaspora, and Acaulospora, whereas relatively high abundances of Dominikia and Pacispora were observed in XYXG. In the soil treatments, the relatively high abundance of Geosiphon in GYXT differed from that in XYXT, which had relatively high abundances of Ambispora, Archaeospora, Funneliformis, and Kamienskia.

3.4.7. Relationship between Soil Environmental Traits and the AMF Community

Soil environmental factors were significantly correlated (p < 0.05) with AMF community structure (Figure 9). The soil environmental factors that significantly affected the AMF species composition in the soils of ancient and adult G. biloba were quite different. In XYXT, Gigaspora was significantly negatively correlated with the TP and TK (p < 0.05), and Dentiscutata was significantly negatively correlated with the TN and TP (p < 0.05). In GYXT, Geosiphon was significantly positively correlated with the TN and TP (p < 0.05), while Sclerocystis was significantly positively correlated with the TN and OM (p < 0.01).

4. Discussion

Ruan et al. [7] reported a complex relationship between G. biloba and soil microbial communities. In the present study, similar to most tree species, bacteria contributed the greatest proportion of the microbial biomass of G. biloba rhizosphere soil, followed by actinomycetes and fungi, and the lowest proportion was that of protozoa. The T-PLFA in the rhizosphere soil is an indicator of microbial community richness. A high total amount represents a rich microbial community structure with a large number of microbial species and populations, which is favorable for nutrient uptake by plants [37]. In the present study, the 19 PLFAs chosen to characterize the soil microbial community structure and biomass were present in the rhizosphere soils of ancient and adult G. biloba, which was inconsistent with hypothesis (1). Moreover, contrary to hypothesis (1), the microbial biomass in the rhizosphere soil of ancient G. biloba was significantly lower than that of adult G. biloba. Previous studies have demonstrated that soil microbial biomass and diversity tend to decrease with increasing tree age, especially for the bacteria and actinomycetes involved in cycling soil substances. In contrast, fungi, which have lower metabolic capabilities, may increase in quantity or in terms of the proportion of the total microbial biomass [38]. The F/B ratio of ancient G. biloba in the present study was slightly higher than that of adult G. biloba, confirming this hypothesis. There is a possibility that the rhizosphere soil of ancient G. biloba may change from a “bacterial” to a “fungal” pattern with the increase of planting time. The F/B ratio reflects the stability of the microbial community; the larger the F/B ratio, the greater the stability [39]. This indicated that the rhizosphere soil microbial community of ancient G. biloba was more stable than that of adult G. biloba. The G+/G− ratio reflects the soil nutrient status, where the higher the value, the higher the nutrient stress [40], suggesting that adult G. biloba trees experience stronger nutrient stress.
Diax et al. [41] concluded that the distribution of the soil microbial population was affected by the combination of local climatic conditions, hydrothermal conditions, soil nutrient status, soil texture, and vegetation composition. According to the correlation analysis between the soil microbial biomass and environmental factors in the present study, soil microorganisms are extremely sensitive to environmental changes, and TP, OM, and pH may be the key factors influencing the soil microbial community in the rhizosphere zone of ancient G. biloba. Tan et al. [42] and Lauber et al. [43] found that changes in the TP content of the soil affected the diversity of the soil bacterial and fungal communities. Accordingly, the significantly lower TP content in ancient G. biloba than in adult G. biloba may be the main reason for the lower microbial biomass in ancient G. biloba. In addition, the soil microbial biomass is related to the soil fertility. The rhizosphere soil of adult G. biloba in this study had a higher organic matter content, and the density of soil bacteria and actinomycetes was also high, which was consistent with the findings of Liu et al. [44]. Although G. biloba has less stringent requirements for soil pH, the metabolic processes of microorganisms have optimal pH ranges. The results of this study suggest that microorganisms in the G. biloba rhizosphere prefer alkaline soils. Therefore, improving the TP, OM, and pH of the soil can increase the microbial biomass in the rhizosphere soil of ancient G. biloba, enhance the microbial community diversity, and promote the absorption of nutrients by ancient trees.
Tong et al. [17] found that inoculation with mycorrhizal fungi could promote the growth of ancient trees and increase soil fertility in practical applications. Therefore, this study examined AMF, which contributes the largest proportion of fungal biomass, in greater depth. Cai et al. [45] also demonstrated that inoculation with AMF treatments also increased the growth and reproduction of phosphate-dissolving bacteria, fungi, and actinomycetes in the soil, but there were significant differences in the increase of the soil microbial population of different AMF. Owing to its ability to generate large amounts of amplicon data, high-throughput sequencing technology is considered one of the most effective molecular techniques for studying community structures and identifying AMF species. It is worth mentioning that this experiment is the first study to employ high-throughput sequencing technology to investigate the diversity of AMF communities in adult G. biloba. This is different from most applications of high-throughput sequencing technology in relation to G. biloba transcriptome, whole genome, and molecular genetic marker discovery [46]. The results indicated the presence of a rich diversity of AMF in G. biloba roots and rhizosphere soil (four orders, ten families, and nineteen genera), which were assigned to 224 virtual taxa. This provides a more comprehensive evaluation than the morphological identification results of nine species from four genera of AMF in G. biloba rhizosphere in Zhejiang Province. Additionally, the present study detected a higher number of ASVs in ancient G. biloba than in adult G. biloba, suggesting closer mutualistic symbiosis between ancient G. biloba and AMF. This aligned with the findings of Pei et al. [47], who reported an increasing trend in the number of AMF operational taxonomic units in the rhizosphere soil of Panax notoginseng with increasing planting years. Interestingly, this contradicted the results of the PLFA analysis, which indicated a significantly lower soil microbial biomass in the rhizosphere of ancient G. biloba than in the rhizosphere soil of adult G. biloba. This suggests that although AMF resources are abundant in the rhizosphere soil of ancient G. biloba, there may be an accumulation of dead but not fully decomposed AMF over long-term growth, leading to a lower biomass of live AMF.
In contrast, in terms of the AMF species composition and community structure, the situation was broadly similar in the roots and rhizosphere soil of ancient and adult G. biloba. The first and second most dominant genera were Glomus and Paraglomus, respectively, in the roots and rhizosphere soils of G. biloba, which was consistent with results reported in different ecosystems by other researchers. Glomus, in particular, is ecologically adaptable, extremely widespread, and a dominant genus in most ecosystems [48,49,50]. Some studies have reported that Paraglomus is the second most dominant genus after Glomus in some studies [51,52]. In contrast, Archaeospora and Ambispora were dominant in the rhizosphere, whereas Diversispora and Claroideoglomus were dominant in the roots. Furthermore, cluster analysis indicated that seven genera had a closer relationship with ancient G. biloba roots, namely Paraglomus, Claroideoglomus, Septoglomus, Sclerocystis, Diversispora, Gigaspora, and Acaulospora, whereas Dominikia and Pacispora had a closer relationship with younger adult G. biloba roots. These findings imply that the application of Diversispora and Claroideoglomus as mycorrhizal fungi in the rhizosphere of ancient G. biloba may improve the mycorrhizal infestation rate.
As evidenced by the aforementioned results, hypothesis (2) is valid. Next, the soil environments of the sampling sites were explored. Differences were found in the effects of different soil factors on AMF. In the rhizosphere soil of adult G. biloba, the results suggest that TN may be the main influencing factor for Acaulospora and Kamienskia, EC may be the main influencing factor for Funneliformis, and TP and TK may be the main influencing factors for Gigaspora and Dentiscutata. Dentiscutata may also be influenced by OM. Geosiphons may be affected by TN and TP in the rhizosphere soil of ancient G. biloba, Geosiphon may be affected by TN and TP, and Sclerocystis may be influenced by TN and OM. Rhizosphere soil factors are important ecological factors that have complex effects on AMF species, diversity, and abundance. However, none of the six soil factors tested had a significant effect on either Claroideoglomus or Diversispora, which can infest the roots of G. biloba. In future research, additional soil factors should be investigated to explore the driving factors and provide a more suitable soil environment.
Furthermore, it will be necessary to isolate and identify the dominant strains in the rhizosphere soil of ancient G. biloba through wet screening, cultivation, and propagation. In addition, the rhizosphere soil should be inoculated with suitable AMF agents to increase the biomass of living AMF in the rhizosphere of ancient trees and improve the mycorrhizal infestation rate and root vigor of ancient G. biloba. This might produce a more obvious effect than the application of exogenous fungi. It is hoped that self-produced mycorrhizal agents obtained from indigenous fungi can reduce the cost of rejuvenation and realize the great potential of using AMF as biofertilizers. This will promote the localized application of suitable AMF to ancient G. biloba, provide more products and choices for the protection and rejuvenation of ancient trees, and play a role in promoting the sustainable protection of ancient trees.

Author Contributions

Conceptualization, J.M. and W.L.; methodology, J.M., F.P., Z.Z., and W.L.; software, J.M. and X.S.; validation, J.M., Y.Y., and Y.W.; formal analysis, J.M.; investigation, J.M., Y.T. and W.L.; resources, Y.T. and W.L.; data curation, J.M. and Y.Y.; writing—original draft, J.M., Q.W., Y.Y., F.P., Z.Z., X.S., and Y.W.; writing—review and editing, Q.W., J.M., F.P., Z.Z. and W.L.; visualization, J.M. and Y.Y.; supervision, Y.T. and W.L.; project administration, Y.T. and W.L.; funding acquisition, Y.T. and W.L. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the Special Fund for Scientific Research of Shanghai Landscaping and City Appearance Administrative Bureau (No. G230505) and the Central Finance Forestry Science and Technology Promotion Demonstration Project (No. JXTG [2024] 25).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The following information was supplied regarding data availability: Data is available at NCBI BioProject accession number PR]NA1142869 (SAMN42966095, SAMN42966096, SAMN42966097, SAMN42966098, SAMN42966099, SAMN42966100, SAMN42966101, SAMN42966102, SAMN42966103, SAMN42966104, SAMN42966105, SAMN42966106, SAMN42966107, SAMN42966108, SAMN42966109, SAMN42966110, SAMN42966111, SAMN42966112, SAMN42966113, SAMN42966114, SAMN42966115, SAMN42966116, SAMN42966117, SAMN42966118, SAMN42966119, SAMN42966120.

Conflicts of Interest

Author Jieying Mao was employed by the company Ningbo Yongneng Biomass Energy Development Co., Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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Figure 1. Sampling positions and process. (a) Map of study sites; (b) sampling sites; (c) sampling process; and (d) sampling diagram.
Figure 1. Sampling positions and process. (a) Map of study sites; (b) sampling sites; (c) sampling process; and (d) sampling diagram.
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Figure 2. Microbial biomass in the rhizosphere soil of Ginkgo biloba. ns indicates p > 0.05, * indicates p < 0.05, ** indicates p < 0.01. The following is the same.
Figure 2. Microbial biomass in the rhizosphere soil of Ginkgo biloba. ns indicates p > 0.05, * indicates p < 0.05, ** indicates p < 0.01. The following is the same.
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Figure 3. Analysis of soil environmental traits and microbial biomass. * indicates p < 0.05, ** indicates p < 0.01.
Figure 3. Analysis of soil environmental traits and microbial biomass. * indicates p < 0.05, ** indicates p < 0.01.
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Figure 4. Arbuscular mycorrhizal fungi (AMF) community composition of the root and rhizosphere soil of Ginkgo biloba at the genus level. XYXG represents the roots of adult Ginkgo biloba, XYXT represents the rhizosphere soil of adult Ginkgo biloba, GYXG represents the roots of ancient Ginkgo biloba, and GYXT represents the rhizosphere soil of ancient Ginkgo biloba.
Figure 4. Arbuscular mycorrhizal fungi (AMF) community composition of the root and rhizosphere soil of Ginkgo biloba at the genus level. XYXG represents the roots of adult Ginkgo biloba, XYXT represents the rhizosphere soil of adult Ginkgo biloba, GYXG represents the roots of ancient Ginkgo biloba, and GYXT represents the rhizosphere soil of ancient Ginkgo biloba.
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Figure 5. Venn diagram of arbuscular mycorrhizal fungi (AMF) community in the root and rhizosphere soil of Ginkgo biloba.
Figure 5. Venn diagram of arbuscular mycorrhizal fungi (AMF) community in the root and rhizosphere soil of Ginkgo biloba.
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Figure 6. Diversity analysis of the arbuscular mycorrhizal fungi (AMF) community in the root and rhizosphere soil of Ginkgo biloba. (a) the chart of Chao1 data analysis; (b) the chart of Shannon analysis; (c) the chart of Simpson analysis; (d) the chart of Pielou’s evenness analysis. Each point in the graph represents the data for each sample.
Figure 6. Diversity analysis of the arbuscular mycorrhizal fungi (AMF) community in the root and rhizosphere soil of Ginkgo biloba. (a) the chart of Chao1 data analysis; (b) the chart of Shannon analysis; (c) the chart of Simpson analysis; (d) the chart of Pielou’s evenness analysis. Each point in the graph represents the data for each sample.
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Figure 7. Principal coordinate analysis (PCoA) of arbuscular mycorrhizal fungi (AMF) community diversity in the root and rhizosphere soil of Ginkgo biloba.
Figure 7. Principal coordinate analysis (PCoA) of arbuscular mycorrhizal fungi (AMF) community diversity in the root and rhizosphere soil of Ginkgo biloba.
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Figure 8. Species clustering heat map of the arbuscular mycorrhizal fungi (AMF) community in the root and rhizosphere soil of Ginkgo biloba.
Figure 8. Species clustering heat map of the arbuscular mycorrhizal fungi (AMF) community in the root and rhizosphere soil of Ginkgo biloba.
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Figure 9. Analysis of soil environmental traits and the arbuscular mycorrhizal fungi (AMF) community. * indicates p < 0.05, ** indicates p < 0.01.
Figure 9. Analysis of soil environmental traits and the arbuscular mycorrhizal fungi (AMF) community. * indicates p < 0.05, ** indicates p < 0.01.
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Table 1. Overview of Ginkgo biloba habitat survey and soil sampling sites.
Table 1. Overview of Ginkgo biloba habitat survey and soil sampling sites.
CategoryNumberTree Age/YearsLongitude and LatitudeSiteGrowth StatusGrowth Environment Characteristics
Ancient G. biloba021425031°23′3″ N, 121°14′46″ ELiuyi CommunitynormalWithin the green area, there is a cluster of ancient trees, surrounded by residential areas and parking lots. There is a considerable amount of construction and household waste in the vicinity.
0215250normal
0217250normal
0343180normal
034518031°20′37″ N, 121°14′11″ EZiqi Donglai ParknormalIn the park, grass is regularly removed under the trees, and the surrounding area is covered with white clover vegetation.
0346180normal
035218031°21′24″ N, 121°14′6″ ENear Shengxin Road Suspension Binhai BridgenormalLakeside area with exposed ground, protected by a fence.
0353180normal
Adult G. bilobaYL15031°9′14′′ N, 121°8′35″ EHua Le RoadnormalAdjacent to the main urban thoroughfare, limited growing space.
YL250normal
YL350normal
YL450normal
YL550normal
Note: The growth status of ancient G. biloba was judged according to the People’s Republic of China LY/T 3073-2018 “Technical regulations for the management and conservation of old and notable trees”. The growth of adult G. biloba was judged by the same industry standards.
Table 2. Characteristic microbes of phospholipid fatty acids (PLFAs).
Table 2. Characteristic microbes of phospholipid fatty acids (PLFAs).
Characterization of Microbial TaxaPLFA Species
Fungi (F)18:1ω9c, 18:2ω6c, and 18:3ω6c
Gram-positive bacteria (G+)i14:0, i15:0, a15:0, i16:0, a17:0, and i17:0
Gram-negative bacteria (G−)16:1ω7c, cy 17:0c, 18: 1ω7c, and cy 19:0c
Actinomycetes (A)10Me16:0, 10Me17:0, and 10Me18:0
Arbuscular mycorrhizal fungi (AMF)16:1ω5c
Protozoa (P)20:3ω6c and 20:4ω6c
Note: i, a, cy, and Me represent isopropyl, anti-isopropyl, cyclopropyl, and methyl-branched fatty acids, respectively; ω and c represent methyl terminal and homeotropic space structures, respectively.
Table 3. Soil chemical analyses of the rhizosphere soil of Ginkgo biloba.
Table 3. Soil chemical analyses of the rhizosphere soil of Ginkgo biloba.
SoilTN (g·kg−1) TP (g·kg−1) TK (g·kg−1) OM (g·kg−1) pHEC (mS·cm−1)
XYXT1.34 ± 0.08 a2.02 ± 0.15 a4.10 ± 0.32 b30.85 ± 3.51 a8.23 ± 0.03 a0.17 ± 0.014 a
GYXT2.00 ± 0.28 a1.39 ± 0.22 b5.78 ± 0.19 a29.29 ± 4.32 a7.79 ± 0.05 b0.13 ± 0.005 b
Note: XYXT represents the rhizosphere soil of adult Ginkgo biloba, and GYXT represents the rhizosphere soil of ancient Ginkgo biloba. Different lowercase letters indicate significant differences (p < 0.05). The following is the same.
Table 4. Soil microbial community structure ratios of Ginkgo biloba.
Table 4. Soil microbial community structure ratios of Ginkgo biloba.
TreatmentsF/BG+/G−cy17:0c/16:1ω7c
XYXT0.173 ± 0.004 a1.346 ± 0.122 a0.599 ± 0.006 a
GYXT0.182 ± 0.004 a1.100 ± 0.027 a0.360 ± 0.012 b
Different lowercase letters indicate significant differences (p < 0.05).
Table 5. Virtual taxa of AMF in the root and rhizosphere soil of Ginkgo biloba obtained via high-throughput sequencing.
Table 5. Virtual taxa of AMF in the root and rhizosphere soil of Ginkgo biloba obtained via high-throughput sequencing.
OrderFamilyGenusVirtual Taxa
GlomeralesGlomeraceaeGlomusG. microaggregatum VTX00104, G. hoi VTX00199
VTX00053, VTX00066, VTX00068, VTX00070, VTX00072–VTX00074, VTX00076, VTX00077, VTX00079, VTX00080, VTX00082–VTX00084, VTX00088, VTX00089, VTX00093, VTX00096, VTX00098, VTX00109, VTX00112, VTX00120–VTX00122, VTX00124–VTX00127, VTX00137, VTX00140, VTX00143, VTX00150, VTX00151, VTX00154, VTX00156, VTX00159, VTX00166, VTX00167, VTX00174, VTX00175, VTX00178–VTX00184, VTX00186, VTX00188, VTX00189, VTX00191, VTX00194, VTX00197, VTX00200, VTX00202, VTX00206, VTX00209, VTX00212, VTX00214, VTX00216, VTX00219, VTX00223, VTX00224, VTX00234, VTX00235, VTX00253, VTX00259, VTX00270, VTX00290–VTX00292, VTX00294, VTX00301, VTX00304, VTX00305, VTX00309, VTX00312, VTX00316, VTX00317, VTX00319, VTX00322–VTX00327, VTX00329, VTX00331, VTX00333, VTX00342–VTX00344, VTX00359, VTX00360, VTX00362, VTX00364, VTX00366, VTX00368–VTX00373, VTX00382–VTX00384, VTX00386–VTX00390, VTX00392, VTX00393, VTX00395, VTX00397–VTX00399, VTX00404, VTX00409, VTX00411–VTX00413, VTX00416, VTX00419, VTX00420, VTX00422, VTX00423, VTX00426, VTX00427, VTX00432, VTX00436, VTX00437, VTX00441–VTX00443, VTX00448, VTX00452, and VTX00453
GlomeralesRhizophagusR. clarus VTX00090, R. proliferus VTX00099, R. intraradices VTX00105, and R. irregularis VTX00114
SeptoglomusS. viscosum VTX00063 and S. constrictum VTX00064
SclerocystisS. sinuosa VTX00069 and S. coremioides VTX00268
FunneliformisF. mosseae VTX00067
DominikiaD. indica VTX00222
KamienskiaK. perpusilla VTX00287
ClaroideoglomeraceaeClaroideoglomusC. lamellosum VTX00193 and C. claroideum VTX00225 and VTX00279
VTX00055–VTX00057, VTX00237, VTX00276–VTX00278, VTX00297, VTX00340, VTX00357, VTX00358, and VTX00402
ParaglomeralesParaglomeraceaeParaglomusP. brasilianum VTX00239, P. laccatum VTX00281, and P. majewskii VTX00335
VTX00001, VTX00002, VTX00308, VTX00337, VTX00348–VTX00352, VTX00375, VTX00433, VTX00435, VTX00444, and VTX00446
ArchaeosporalesAmbisporaceaeAmbisporaA. leptoticha VTX00242, A. fennica VTX00283, A. granatensis VTX00339
VTX00405
ArchaeosporalesArchaeosporaceaeArchaeosporaVTX00004–VTX00009, VTX00051, VTX00338, VTX00376, and VTX00450
GeosiphonaceaeGeosiphonG. pyriformis VTX00241
DiversisporalesDiversisporaceaeDiversisporaD. epigaea VTX00061
VTX00040, VTX00306, VTX00354–VTX00356, VTX00380, and VTX00401
RedeckeraR. fulvum VTX00262
AcaulosporaceaeAcaulosporaA. spinosa VTX00026 and A. longula VTX00028
VTX00013, VTX00015, VTX00016, VTX00227, VTX00231, VTX00272, and VTX00328
GigasporaceaeScutellosporaS. dipurpurescens VTX00049, S. nodosa VTX00052, S. spinosissima VTX00254, S. projecturata VTX00260, and S. nodosa VTX00261
VTX00318
GigasporaG. decipiens VTX00039
DentiscutataD. heterogama VTX00255
PacisporaceaePacisporaP. scintillans VTX00284
VTX00011
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MDPI and ACS Style

Mao, J.; Wang, Q.; Yang, Y.; Pan, F.; Zou, Z.; Su, X.; Wang, Y.; Liu, W.; Tang, Y. A Treasure Trove of Urban Microbial Diversity: Community and Diversity Characteristics of Urban Ancient Ginkgo biloba Rhizosphere Microorganisms in Shanghai. J. Fungi 2024, 10, 720. https://doi.org/10.3390/jof10100720

AMA Style

Mao J, Wang Q, Yang Y, Pan F, Zou Z, Su X, Wang Y, Liu W, Tang Y. A Treasure Trove of Urban Microbial Diversity: Community and Diversity Characteristics of Urban Ancient Ginkgo biloba Rhizosphere Microorganisms in Shanghai. Journal of Fungi. 2024; 10(10):720. https://doi.org/10.3390/jof10100720

Chicago/Turabian Style

Mao, Jieying, Qiong Wang, Yaying Yang, Feng Pan, Ziwei Zou, Xiaona Su, Yi Wang, Wei Liu, and Yaohua Tang. 2024. "A Treasure Trove of Urban Microbial Diversity: Community and Diversity Characteristics of Urban Ancient Ginkgo biloba Rhizosphere Microorganisms in Shanghai" Journal of Fungi 10, no. 10: 720. https://doi.org/10.3390/jof10100720

APA Style

Mao, J., Wang, Q., Yang, Y., Pan, F., Zou, Z., Su, X., Wang, Y., Liu, W., & Tang, Y. (2024). A Treasure Trove of Urban Microbial Diversity: Community and Diversity Characteristics of Urban Ancient Ginkgo biloba Rhizosphere Microorganisms in Shanghai. Journal of Fungi, 10(10), 720. https://doi.org/10.3390/jof10100720

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