长期施肥对棕壤丛枝菌根真菌群落结构及其侵染的影响

doi:10.3864/j.issn.0578-1752.2018.24.008

摘要/Abstract

摘要：

目的丛枝菌根(Arbuscular mycorrhizal, AM)真菌有改善根际土壤环境、促进植物对养分的吸收、增强植物抗逆性和增加农作物产量等重要作用。本研究旨在探明长期施肥条件下玉米-大豆轮作棕壤丛枝菌根真菌群落结构、对玉米根系侵染的变化及其影响因素。方法以沈阳农业大学棕壤肥料长期定位试验（38年）耕层（0—20 cm）土壤为材料,于2016年6月选取其中6个施肥处理：（1）不施肥处理（CK）;（2）单施化学氮肥（N）;（3）施用化学氮磷肥（NP）;（4）施用化学氮磷钾肥（NPK）;（5）单施有机肥（M）;（6）有机肥和化学氮磷肥配施（MNP）,采用PCR-DGGE、克隆测序及台盼蓝染色法,分析土壤和玉米根系定殖的AM真菌群落结构及侵染率,并结合环境因素进行冗余分析和典型对应分析。结果施用有机肥处理土壤的碱解氮（AHN）、速效磷（AP）、速效钾（AK）、铵态氮（NH₄ ⁺-N）、硝态氮（NO₃ ^--N）和可溶性有机碳（DOC）含量显著高于单施化肥和不施肥处理,且趋势为：有机肥处理>化肥处理>不施肥处理;与不施肥处理相比,单施化肥处理显著降低了土壤pH值,而施用有机肥处理显著提高了土壤pH值。通过PCR-DGGE及割胶测序,从土壤中得到AM真菌条带22条,根系AM真菌条带仅9条,共分离出13个OTU,从土壤样品中分离的AM真菌种群主要为球囊霉菌和巨孢囊霉属,而侵染玉米根系的AM真菌只有球囊霉菌。聚类分析表明长期不同施肥将棕壤中AM真菌分为了三大类群,分别为单施氮肥处理、施用有机肥处理和其他处理;根系AM真菌分为三大类群,第一类群NPK处理、第二类群为M处理和NP处理、第三类群为其他施肥处理。施用有机肥处理AM真菌的孢子密度显著高于单施化肥和不施肥处理,趋势为：有机肥处理﹥化肥处理﹥不施肥处理。各施肥处理AM真菌侵染率趋势为：NPK处理>施用有机肥处理>其他施肥处理。冗余分析结果表明棕壤AM真菌多样性与土壤理化性质无相关性,而孢子密度与土壤AHN、NH₄ ⁺-N、AP、AK、DOC及土壤含水量呈显著正相关;侵染率与土壤NO₃ ^--N呈显著正相关;侵染率与孢子密度之间呈显著正相关;AM真菌的多样性与孢子密度和侵染率之间没有相关性。典型对应分析表明AHN、AK、DOC、 NH₄ ⁺-N 对AM真菌的群落组成影响显著。结论长期施肥通过改变土壤理化性质,从而对棕壤AM真菌的群落结构产生了显著影响,进而对AM真菌的侵染产生影响。

关键词: 丛枝菌根真菌, 棕壤, 长期施肥, 群落结构, 侵染率

Abstract:

【Objective】AM (Arbuscular mycorrhizal) fungi plays important roles like improving the rhizosphere soil environment, promoting the uptake of nutrients by plants, enhancing resistance of plant, and increasing crop yield and quality. This study aimed to explore changes of community structures and colonization rate of AM fungi and to find out the main factors which affected the changes under a corn-soybean rotation system and a long-term fertilization for 38 years in a brown soil. 【Method】 Soil samples (0-20 cm) were taken from the six treatments of the long-term fertilization experiment in June 2016, including (1) no fertilizer (CK); (2) chemical N input (N); (3) chemical N and P input (NP); (4) chemical N, P and K input (NPK); (5) pig manure (M); (6) pig manure, chemical N and P (MNP). Then the soil samples were analyzed by using PCR-DGGE, gel-recovery, sequencing and trypan blue staining. Relationship between community and colonization rate of AM fungi and environmental factors were analyzed by Redundancy analysis and Canonical Correlation analysis. 【Result】 The result showed that the contents of alkali-hydrolysable nitrogen (AHN), available phosphorus (AP), available potassium (AK) , ammonium nitrogen (NH₄ ⁺-N), nitric nitrogen (NO₃ ^--N) and dissolved organic carbon (DOC) under organic fertilization treatments were significantly higher than them under the CK treatment and chemical fertilization treatments, and the trend was: organic fertilizer treatments > chemical fertilizer treatments > CK treatment. Compared with the CK treatment, soil pH was decreased in chemical fertilizer treatments and increased in organic fertilizer treatments. 22 bands of AM fungi from soil and 9 bands of AM fungi from root were obtained by gel-recovery, and 13 OTU were obtained by BLAST. The result of sequencing showed that AM fungi species isolated from soil samples were mainly Glomeromycota and Gigasporaceae, while infected AM fungi was only Glomeromycota. The cluster analysis showed that community structures of soil AM fungi were divided into three groups under a long-term fertilization in a brown soil, namely N treatment, organic fertilizer treatments and another fertilizer treatments. Community structures of infected AM fungi were also divided into three groups, namely NPK treatment, M treatment and NP treatment, and another fertilizer treatments. The spore density of AM fungi under organic fertilizer treatments was significantly higher than that under chemical fertilizer treatments and non-fertilizer treatment, and the trend was: organic fertilizer treatments > chemical fertilizer treatments > CK treatment. The trend of colonization rate of AM fungi under different fertilizer treatments was: NPK treatment > organic fertilizer treatments > another fertilizer treatments. Redundancy analysis showed that spore density was positively correlated with soil AHN, NH₄ ⁺-N, AP, AK, DOC and soil moisture content, and colonization rate was positively correlated with nitric nitrogen content. The colonization rate was positively correlated with spore density, while diversity index of AM fungi was neither correlation with colonization rate nor with spore density. Canonical Correlation analysis showed AHN, AK, DOC and NH₄ ⁺-N significantly influenced the ribotypes of AM fungi. 【Conclusion】 The long-term fertilization changed community structures of AM fungi by changing the physicochemical properties of the soil, and then affected colonization of AM fungi.

Key words: Arbuscular mycorrhizal fungi, brown soil, long-term fertilization, community structure, colonization rate

郭静,罗培宇,杨劲峰,李冬冬,黄月玥,韩晓日. 长期施肥对棕壤丛枝菌根真菌群落结构及其侵染的影响[J]. 中国农业科学, 2018, 51(24): 4677-4689.

GUO Jing,LUO PeiYu,YANG JinFeng,LI DongDong,HUANG YueYue,HAN XiaoRi. Influence of Long-term Fertilization on Community Structures and Colonization of Arbuscular mycorrhizal Fungi in a Brown Soil[J]. Scientia Agricultura Sinica, 2018, 51(24): 4677-4689.

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链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2018.24.008

https://www.chinaagrisci.com/CN/Y2018/V51/I24/4677

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引物名称Primer	引物序列（5′-3′）Primer sequence（5′-3′）	反应条件Conditions
NS1	GTAGTCATATGCTTGTCTC	94℃预变性3 min;94℃ 1 min,50 ℃ 1 min,72 ℃ 1 min,30个循环;72 ℃ 7 min (一轮) An initial denaturation of 94℃for 3 min; 94℃ 1 min, 50℃ 1 min, 72℃ 1 min, 30 cycles; 72℃ 7 min (The first round)
NS41	CCCGTGTTGAGTCAAATTA
GC-NS31	TTGGAGGGCAAGTCTGGTGCC
AM1	GTTTCCCGTAAGGCGCCGAA	94 ℃预变性3 min;94 ℃ 30 s,58 ℃ 45 s,72 ℃ 45 s,28个循环;72 ℃ 7 min（二轮） An initial denaturation of 94℃ for 3 min; 94℃ 30 s, 58℃ 45 s, 72℃ 45 s, 28 cycles; 72℃ 7 min (The second round)
GC-ARCH1311	TGCTAAATAGCCAGGCTGY
NS8	TCCGCAGGTTCACCTACGGA

处理 Treatment	pH (H₂O)	速效磷 AP (mg·kg^-1)	速效钾 AK (mg·kg^-1)	碱解氮 AHN (mg·kg^-1)	硝态氮 NO₃^--N (mg·kg^-1)	铵态氮 NH₄⁺-N (mg·kg^-1)	可溶性有机碳 DOC (mg·kg^-1)	土壤含水量 SM (%)
CK	5.75±0.01b	1.19±0.64d	96.66±1.21c	85.04±0.68f	12.06±0.02e	3.97±0.18d	39.42±0.26e	15.40±0.14b
N	4.87±0.00d	2.14±0.64d	81.62±1.86c	110.43±0.94d	48.38±1.03c	4.45±0.12cd	65.41±0.05d	15.41±0.22b
NP	5.35±0.01c	16.15±0.22c	76.94±2.32c	96.60±1.17e	51.22±1.39bc	4.83±0.16bc	71.29±0.74c	15.12±0.38b
NPK	4.99±0.01c	17.10±0.63c	107.02±1.16c	120.73±2.43c	55.97±0.74b	5.26±0.046b	73.10±0.17c	15.80±0.04b
M	6.64±0.06a	137.72±2.29b	209.96±9.98b	132.54±1.74b	41.81±0.75d	5.33±0.09b	108.44±2.51b	18.11±0.04a
MNP	5.78±0.02b	182.15±7.01a	268.12±9.75a	165.56±2.24a	73.63±0.43a	7.75±0.12a	126.45±1.30a	18.20±0.73a

处理 Treatment	香农指数 Shannon-Wiener(H)	均匀度 Evenness (E)	条带数 Bands
CK	2.04±0.01c	0.982±0.00a	8
N	1.93±0.01d	0.988±0.01a	7
NP	2.18±0.02a	0.991±0.01a	9
NPK	1.78±0.03e	0.997±0.02a	6
M	2.09±0.05b	0.999±0.01a	8
MNP	1.78±0.02e	0.995±0.01a	6

处理 Treatment	香农指数 Shannon-Wiener(H)	均匀度 Evenness(E)	条带数 Bands
CK	0.88±0.06c	0.799±0.02b	3
N	1.03±0.09c	0.740±0.01b	4
NP	1.91±0.03a	0.981±0.01a	7
NPK	1.92±0.02a	0.985±0.01a	7
M	1.91±0.01a	0.984±0.01a	7
MNP	1.36±0.01b	0.988±0.01a	4