简介概要

长期铬污染对土壤真菌群落的影响

来源期刊:中国有色金属学报(英文版)2018年第9期

论文作者:胡瑾 孟德龙 刘学端 梁伊丽 尹华群 刘宏伟

文章页码:1838 - 1846

关键词:真菌群落;α多样性;β多样性;铬污染;高通量测序

Key words:fungal community; alpha-diversity; beta-diversity; Cr contamination; high-throughput sequencing

摘    要:为进一步研究重金属污染生态系统中真菌群落的变化,以废弃铬盐厂污染的土壤为研究对象,通过二代测序ITS扩增子技术,研究真菌微生物群落。结果显示,铬污染虽然改变了真菌群落的组成和结构,但是对其多样性并没有显著的影响。不同种类的真菌对铬污染的响应不同。LEfSe分析结果显示,铬污染环境会导致真菌种类的变化,这是由于高浓度的铬污染能够对真菌细胞直接产生毒性,或者通过改变土壤性质,使真菌细胞对土壤中碳、氮等能源物质的利用能力下降。在所有铬的形态中,有机结合态和可交换态的铬对真菌群落的影响最显著;而土壤性质中,有机质对真菌群落的影响最为显著。

Abstract: To further study the fungal community in heavy metal contaminated ecosystems, soil samples were collected from an abandoned chromium (Cr) factory, and fungal community was analyzed by Illumina sequencing of Internal Transcribed Spacer (ITS) amplicons. The results showed that Cr contamination changed the composition and structure of soil fungal community, but didn’t change the diversity. Fungus showed various responses to Cr contamination. LEfSe analysis revealed that the biomarker changed a lot in the Cr-contaminated samples in comparison with that in the control samples. The changes in fungal community may be caused by the direct toxic effects on fungi by high concentration of Cr and the significant change in soil properties resulting from Cr contamination. Among all the Cr fractions, organic matter-bound Cr and exchangeable Cr showed significant effects on the fungal community and organic matter also showed a significant effect on soil fungal community.



详情信息展示

Trans. Nonferrous Met. Soc. China 28(2018) 1838-1846

Response of soil fungal community to long-term chromium contamination

Jin HU1,2, De-long MENG1,2, Xue-duan LIU1,2, Yi-li LIANG1,2, Hua-qun YIN1,2, Hong-wei LIU1,2

1. School of Minerals Processing and Bioengineering, Central South University, Changsha 410083, China;

2. Key Laboratory of Biometallurgy, Ministry of Education, Central South University, Changsha 410083, China

Received 2 December 2017; accepted 29 May 2018

Abstract: To further study the fungal community in heavy metal contaminated ecosystems, soil samples were collected from an abandoned chromium (Cr) factory, and fungal community was analyzed by Illumina sequencing of Internal Transcribed Spacer (ITS) amplicons. The results showed that Cr contamination changed the composition and structure of soil fungal community, but didn’t change the diversity. Fungus showed various responses to Cr contamination. LEfSe analysis revealed that the biomarker changed a lot in the Cr-contaminated samples in comparison with that in the control samples. The changes in fungal community may be caused by the direct toxic effects on fungi by high concentration of Cr and the significant change in soil properties resulting from Cr contamination. Among all the Cr fractions, organic matter-bound Cr and exchangeable Cr showed significant effects on the fungal community and organic matter also showed a significant effect on soil fungal community.

Key words: fungal community; alpha-diversity; beta-diversity; Cr contamination; high-throughput sequencing

1 Introduction

Chromium (Cr) is a chemical element that has been widely applied in many areas such as chemical, factories and foundry. About 85% of the Cr is used in metal alloys [1,2]. The remaining Cr compounds at abandoned industrial sites have high toxic impacts on microorganism, animals and plants [3]. The Cr is an essential micro-nutrient of animals and humans, but high Cr concentration has great harm to animals and humans and causes serious diseases, such as cancer and deformity, or even leads to death. Meanwhile, no positive effects of Cr have been found in plants [4]. CERVANTES et al [4] found that both 0.5 mg/L Cr(VI) solution and 5 mg/L Cr(VI) in soil were harmful to plants. Cr also has great diffusion capacity, because hexavalent chromium, a highly toxic form of Cr, is highly water-soluble and mobile [5]. Soluble Cr can cross the plasma membrane and enter into organisms. The generated free Cr radicals cause direct DNA alterations or other toxic effects [6,7]. CHAI et al [8] have proven that indigenous bacteria in soils could deal with Cr contamination effectively.

Soil microbes are important indicators of soil quality, and heavy metal contamination causes significant changes in the microbial community. Many studies have focused on the responses of bacteria to heavy metal pollution [9-12]. For example, CHAI et al [8] found that the bacteria strains isolated from Cr contaminated soils showed obvious Cr resistance and had effective Cr reduction ability, and our recent study [13] indicated that microbial communities adapt to heavy metal contamination through complex interactions. The fungal community is also an important part of the below-ground ecosystem as the fungi are important organic matter decomposer. Fungal communities cause various responses to heavy metal contamination. For example, VAL et al [14] and LIAO et al [15] found that fungi community composition changed drastically in response to the heavy metals.

However, there are few studies focused on the fungal community in soils facing serious and long-term heavy metal pollution (e.g. the fungal community in soil below the Cr slag). Whereas, understanding the response of fungal community to long-term Cr contamination is of fundamental importance for bioremediation of Cr pollutions.

In the present work, we sampled long-term Cr contaminated soils from an abandoned Cr factory, and investigated soil fungal community using ITS Illumina Miseq sequencing. The aims of this work were to investigate (1) how fungal community responded to the long-term Cr-contamination and (2) which Cr fraction(s) was the major factors affecting fungal community.

2 Experimental

2.1 Site description and soil sampling

Soil samples were gathered in October, 2015. Sampling site is at an abandoned chromium (Cr) salt factory located in Hunan Province, China (E112°58'0", N28°16'23"). The chromate plant has been abandoned for 13 years and no plants are growing in the polluted area. Soil samples (contaminated) were collected (0-20 cm) under the chromate slag heap; meanwhile, control soils were sampled from the adjacent area around the Cr deposit for the same original parent material with contaminated soils (about 500 m far away from the contaminated site and plants can grow). Each type of soil (control and contaminated) was collected three times as the biological replicates. All soil samples were separated into two parts. One part was frozen in liquid nitrogen and stored at -80 °C for further molecular analysis, and the other was brought into the library and was stored at 4 °C for physiochemical property analysis.

Soil physiochemical properties including pH, organic matter and total nitrogen are shown in Table 1, and the Cr contents including exchangeable Cr, Fe/Mn oxides-bound Cr and organic matter-bound Cr were measured according to methods by RAURET et al [16] and MILLER and ZITTEL [17]. Briefly, the exchangeable Cr was extracted using acetate solution, the Fe/Mn oxides-bound Cr was extracted using NH4OH·HCl and HNO3 solution and the organic matter-bound Cr was extracted using H2O2 and NH4OAc solution. The concentration of extracted Cr was measured by the 1,5-diphenylcarbohydrazide spectro- photometric method.

Fungal colony form units (CFU) were determined as described by SIEUWERTS et al [18].

2.2 DNA extraction, PCR and sequencing

DNA was extracted from 10 g soils by DNA extraction method [19]. Then, the quantity and quality of extracted DNA were checked by using a NanoDrop ND-100 spectrophotometer and agarose gel electrophoresis.

PCR amplification was performed on Applied Biosystems 2720 Thermal Cycler (ABI Inc., USA) using ITS primer pair gITS7F (5’-GTG ART CAT CGA RTC TTT G-3’) and ITS4R (5’-TCC TCC GCT TAT TGA TAT GC -3’) together with barcodes and Illumina adapter sequences. Amplification system included 0.5 μL of Taq polymerase (TaKaRa, Japan), 5 μL of 10× PCR buffer, 1.5 μL of dNTP mix, 1.5 μL each primer (10 μmol/L, forward and reverse), 2 μL of DNA template (~20 ng/μL) and 38 μL of deionised H2O. The PCR program was configured as follows: denaturation at 94 °C for 5 min, and 35 cycles of 94 °C for 20 s, 57 °C for 25 s, and 68 °C for 45 s, with a final extension at 68 °C for 10 min. PCR products were purified using E.Z.N.A. TM Gel Extraction Kit (OMEGA Bio-tek Inc., Doraville, GA, USA). Then, the concentration and quality of recovered DNA were evaluated by NanoDrop ND-100 Spectrophotometer (NanoDrop Technologies, Wilmington, USA). For constructing the sequencing library, 200 ng of each purified DNA product was mixed together, and the sequencing was performed on Illumina Miseq platform (Illumina Inc., San Diego, CA, USA).

2.3 Data processing and statistical analysis

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