Fungal Community in Soil of Wulongjiang Wetlands Analyzed Using PCR-DGGE
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摘要: 为了解乌龙江湿地土壤真菌群落结构组成及动态变化，本研究通过稀释平板法对湿地土壤可培养真菌数进行测定，并采用变性梯度凝胶电泳（DGGE）技术分析乌龙江湿地土壤真菌群落结构。平板计数结果表明，不同时间不同采样点可培养真菌数的变化幅度较小，每克干土中可培养真菌数介于0.13×104~8.26×104 CFU。真菌18S rDNA的PCR-DGGE图谱显示，每个泳道条带的位置、数目和亮度各有不同，说明不同采样时间不同采样点土壤真菌群落结构存在着差异。DGGE图谱条带多样性分析结果表明，2009年3月、2009年11月和2010年1月所采集土壤样品的真菌群落多样性较其他时间更为丰富；从采样点来看，真菌群落丰富度则表现为样品L >样品M >样品R。对DGGE主要条带和差异性条带进行克隆测序后发现，乌龙江湿地土壤主要真菌类群为子囊菌Ascomycota和毛霉菌Mucoromycota。Abstract: Composition and dynamics of the fungal community in soil of Wulongjiang wetlands were studied. The classical plate count and the denatured gradient gel electrophoresis (DGGE) were applied to determine the cultivable population and diversity of the community. The plate counts of the samples averaged between 0.13×104 and 8.26×104 CFU per g of dry soil with no significant difference due to the locations or time of collection. On the other hand, the PCR-DGGE patterns of 18S rDNA (V1+V2) fragments exhibited variations on the positions, number and lightness of the bands from different lanes, indicating compositional differences among the fungal populations. The DGGE bands digitally analyzed by Quantity One software and the derived Shannon-Wiener index, evenness and abundance showed greater diversities in the samples obtained in March 2009, September 2009 and January 2010 than other times of a year. They also unveiled the differences in abundance of Sample L > Sample M > Sample R. The dominant fungi in the soil were identified to be Ascomycota and Mucoromycota according to a sequence analysis on the DGGE bands.
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Key words:
- Wulongjiang wetland /
- soil fungi /
- community composition /
- PCR-DGGE /
- diversity analysis
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图 1 乌龙江湿地土壤真菌18S rDNA(V1+V2)区扩增片段的DGGE分析
Figure 1. DGGE patterns of 18S rDNA(V1+V2) fragments of fungi in Wulongjiang wetland soil
图 2 乌龙江湿地土壤真菌18S rDNA序列系统发育图谱
Figure 2. Phylogenetic tree on 18S rDNA sequences of fungi in Wulongjiang wetland soil
表 1 乌龙江湿地土壤可培养真菌数量的动态变化
Table 1. Dynamics of culturable fungal population in Wulongjiang wetland soil
[单位/×104CFU·g-1(dry soil)] 样品名称 采样时间/(年-月) 2009-03 2009-05 2009-07 2009-09 2009-11 2010-01 R-1 6.18±0.81 4.32±0.55 4.43±0.12 5.94±0.43 4.04±0.43 8.26±0.28 R-2 2.68±0.44 2.50±0.80 4.09±0.77 1.99±0.14 1.93±0.36 4.96±0.51 R-3 1.02±0.03 8.62±0.18 1.73±0.22 2.75±0.62 1.60±0.81 2.80±0.84 M-1 6.03±0.54 3.73±0.86 2.45±0.35 2.75±0.63 1.63±0.23 0.59±0.12 M-2 2.41±0.46 1.34±0.18 3.51±0.76 3.57±0.23 4.50±0.99 4.44±0.23 M-3 1.95±0.31 1.84±0.07 2.24±0.41 1.87±0.19 4.73±0.61 2.19±0.51 L-1 2.27±0.45 0.29±0.06 0.84±0.58 0.50±0.01 1.14±0.18 0.88±0.16 L-2 2.11±0.19 0.56±0.11 2.00±0.71 0.19±0.09 0.90±0.05 0.33±0.08 L-3 1.24±0.15 1.66±0.44 1.91±0.76 0.23±0.04 2.49±0.17 0.13±0.01 
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表 2 DGGE图谱的Shannon-Wiener指数、均匀度和丰富度分析结果
Table 2. Shannon-Wiener index, evenness and abundance derived from DGGE patterns
采样时间/(年-月) 采样点 Shannon-Wiener指数 均匀度 丰富度 2009-03 R 3.18 0.97 27 M 3.13 0.97 25 L 3.18 0.98 26 2009-05 R 2.75 0.97 17 M 2.68 0.95 17 L 2.87 0.94 21 2009-07 R 3.04 0.97 23 M 2.75 0.97 17 L 2.88 0.96 20 2009-09 R 2.92 0.95 22 M 2.95 0.97 21 L 2.79 0.97 18 2009-11 R 3.08 0.98 23 M 3.18 0.97 26 L 3.23 0.98 27 2010-01 R 3.13 0.97 25 M 3.34 0.98 30 L 3.44 0.98 34
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表 3 DGGE代表性条带的比对结果
Table 3. Genomic sequences of dominant DGGE bands obtained by sequencing and BLAST analysis
编号 最相似菌株 登录名 相似率/% 最相似菌群 Band 1 Hyphochytrium catenoides X80344.1 99 Stramenopiles Band 2 Mortierella alpina AY550125.1 99 Mucoromycota Band 3 Uncultured fungus JN166410.1 99 Fungi Band 4 Mortierella alpina AY546098.1 99 Mucoromycota Band 5 Pseudombrophila guldeniae DQ063001.1 98 Ascomycota Band 6 Pithya cupressina JX268559.1 96 Ascomycota Band 7 Alternaria sp. GE KJ489375.1 100 Ascomycota Band 8 Uncultured archaeospora KT923272.1 100 Mucoromycota Band 9 Fusarium oxysporum KJ126877.1 99 Ascomycota Band 10 Metarhizium anisopliae AF487276.1 99 Ascomycota Band 11 Vischeria stellata KY271662.1 99 Stramenopiles Band 12 Uncultured magnaporthe KF258907.1 96 Ascomycota Band 13 Knufia sp. CBS 268.34 AF346419.1 99 Ascomycota Band 14 Liochthonius sp. AD1300 JQ000035.1 93 Arthropoda Band 15 Graphium fructicola AB007659.1 93 Ascomycota Band 16 Dicyrtomina environmental sample KF258916.1 95 Arthropoda Band 17 Glyphium elatum DQ133009.1 99 Ascomycota Band 18 Poduridae environmental sample EF024377.1 95 Arthropoda Band 19 Sminthuridae environmental sample EF024291.1 96 Arthropoda
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