不同连作年限怀牛膝根际芽孢杆菌和镰刀菌的多样性分析

陈婷; 王娟英; 吴林坤; 陈军; 吴红淼; 林生; 林文雄

doi:10.19303/j.issn.1008-0384.2020.11.009

不同连作年限怀牛膝根际芽孢杆菌和镰刀菌的多样性分析

doi: 10.19303/j.issn.1008-0384.2020.11.009

陈婷^{1, 2,},
王娟英^{1, 2},
吴林坤^{1, 2},
陈军^{1, 2},
吴红淼^{1, 2},
林生^{1, 2},
林文雄^{1, 2, ,}

1.
福建农林大学生命科学学院，福建　福州　350002
2.
福建农林大学福建省农业生态过程与安全监控重点实验室，福建　福州　350002

基金项目: 福建省教育厅中青年教师科研项目（JAT170200）；闽台作物特色种质创制与绿色栽培协同创新中心（2015-75）

详细信息

作者简介:
陈婷（1981−），女，博士，助理研究员，研究方向：农业生态学（E-mail：iamchenting@126.com）

通讯作者:
林文雄（1957−），男，博士，教授，研究方向：农业生态学（E-mail：wenxiong181@163.com）

中图分类号: S 567
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出版历程
- 收稿日期: 2020-07-28
- 修回日期: 2020-09-27
- 网络出版日期: 2020-11-13
- 刊出日期: 2020-11-30

Diversity of Bacillus and Fusarium Species in Rhizosphere Soil under Continuous Achyranthes bidentata Monoculture

CHEN Ting^{1, 2
,},
WANG Juanying^{1, 2},
WU Linkun^{1, 2},
CHEN Jun^{1, 2},
WU Hongmiao^{1, 2},
LIN Sheng^{1, 2},
LIN Wenxiong^{1, 2
, ,}

1.
College of Life Science, Fujian Agriculture and Forestry University, Fuzhou, Fujian 350002, China
2.
Fujian Provincial Key Laboratory of Agroecological Processing and Safety Monitoring, Fuzhou, Fujian　350002, China

摘要

摘要: 目的针对药用植物怀牛膝耐连作甚至表现连作促进的特殊现象，分析不同连作年限怀牛膝根际土壤关键微生物群落结构变化，探讨不同连作年限怀牛膝根际微生物组的演变过程，为破解大多数药用植物连作障碍提供理论借鉴。方法本研究以连作1年、10年、15年怀牛膝根际土壤与撂荒田对照土壤为试验材料，采用变性梯度凝胶电泳（DGGE）技术分析芽孢杆菌属和镰刀菌属群落结构差异。结果在怀牛膝根际土壤中，芽孢杆菌属种类丰富，其中优势菌群为枯草芽孢杆菌（Bacillus stubtilis）和蜡样芽孢杆菌（Bacillus cereus）；且与1年土壤相比，连作10年和15年的土壤中会明显增加枯草芽孢杆菌和耐盐芽孢杆菌的相对含量。镰刀菌属的条带数目相对较少，种类多样性少，在群落结构上CK和15Y比较相似，1年和10年相似，优势菌群为腐皮镰刀菌（Fusarium solani）和尖孢镰刀菌（Fusarium oxysporum）。qPCR定量分析显示，怀牛膝连作下会增加其有益细菌的含量，而有害菌镰刀菌属基本维持在一个水平。结论多年连作怀牛膝能够增加根际有益微生物群落的多样性与种群丰度，抑制病原微生物群落的多样性与种群丰度，通过选择性促抑影响根际微生物组成而自我塑造健康的根际微生态环境。
- 怀牛膝 /
- 连作 /
- 芽孢杆菌 /
- 镰刀菌 /
- DGGE
Abstract: Objective Changes on the key microbial communities in the continuously cropped rhizosphere soil (CCRS) of Achyranthes bidentata Blume were studied to analyze the allelopathic effect for operational improvement on cultivation of the medicinal plants. Method The community structures of Bacillus spp. and Fusarium spp. in the rhizosphere soil of A. bidentata under 1, 10, and 15 years of consecutive monoculture were determined using denaturing gradient gel electrophoresis (DGGE). Soil sample from a virgin land was used as control (CK). Result Abundant Bacillus spp. dominated by B. stubtilis and B. cereus were isolated from the CCRS specimens. The CCRS under 10-year (10Y) and 15-year (15Y) of continuous cropping had a greater relative content of B. subtilis and B. halodurans than their 1-year (1Y) counterpart. With respect to Fusarium spp., the number of bands and species were relatively few, the community structure tended to be similar between CK and 15Y as well as between 1Y and 10Y, and F. solani and F. oxysporum being the dominant species. The qPCR results also indicated that continuous monoculture of A. bidentata increased the abundance of the beneficial bacteria but kept that of the harmful Fusarium spp. largely unchanged. Conclusion Continuous cropping A. bidentata enhanced the diversity and abundance of the beneficial Bacillus spp. without encouraging the expansion of the pathogenic Fusarium community in the rhizosphere. It appeared that the rhizosphere allelopathy under the cultivation practice had created a healthy ecosystem that promoted the growth of A. bidentata.
- Achyranthes bidentata Blume /
- continuous monoculture /
- Bacillus /
- Fusarium /
- DGGE

HTML全文

图 1 土壤总DNA提取（A）、芽孢杆菌属特异片段扩增（B）、镰刀菌属特异片段扩增（C）

注：图中M、1、2、3、4分别表示Marker、对照土壤（CK）、连作1年（1Y）土壤、10年（10Y）土壤、15年（15Y）土壤。

Figure 1. Extracted DNA of A. bidentata from soil (A) and PCR amplifications of Bacillus (B) and Fusarium (C)

Note: Lane M, 1, 2, 3, and 4 represented marker, CK, 1Y, 10Y, and 15Y, respectively.

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图 2 不同连作年限下怀牛膝根际土壤芽孢杆菌属群落的DGGE图谱

注：CK，对照土壤；1Y：连作1年土壤；10Y：连作10年土壤；15Y：连作15年土壤。图中所标识的条带为差异条带。图4同。

Figure 2. DGGE profiles of Bacillus amplified from CK, 1Y, 10Y, and 15Y specimens

Note: In soil specimens of CK, 1Y, 10Y, and 15Y. Differential bands are identified. Same for Fig. 4.

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图 3 芽孢杆菌属DGGE主成分分析

Figure 3. Principal component analysis on DGGE of Bacillus

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图 4 不同连作年限下怀牛膝根际镰刀菌属群落的DGGE图谱

Figure 4. DGGE profiles of amplified Fusarium from CK, 1Y, 10Y, and 15Y specimens

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图 5 镰刀菌属DGGE主成分分析

Figure 5. Principal component analysis on DGGE of Fusarium

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图 6 不同连作年限下怀牛膝根际芽孢杆菌和尖孢镰刀菌绝对定量

Figure 6. Quantification of Bacillus spp. and F. oxysporum in soil specimens

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表 1 PCR 扩增所用引物及程序

Table 1. Primers and program applied for PCR amplification

引物 Primer	序列（5′-3′） Sequence (5′-3′)	PCR程序 PCR program	参考文献 References
Bacillus
R1378	CGGTGTGTACAAGGCCCGGGAACG	94 ℃预变性5 min；35个循环程序：94 ℃，1 min，65 ℃，30 s，72 ℃，1 min；最后72 ℃延伸10 min。 Pre-denaturation at 94 ℃ for 5 min; 35 cycles program: 94 ℃, 1 min, 65 ℃, 30 s, 72 ℃, 1 min; finally extension at 72 ℃ for 10 min.	Drigo, et al. 2009^[19]
Bacf	GGGAAACCGGGGCTAATACCGGAT		Drigo, et al. 2009^[19]
F968-GC	AACGCGAAGAACCTTAC	94 ℃预变性5 min；各2个循环程序：94 ℃，1 min，63 ℃/61 ℃/59 ℃/57 ℃/55 ℃，1 min，72 ℃，1 min，20个循环程序：94 ℃，1 min，55 ℃，1 min，72 ℃，1 min；最后72 ℃延伸10 min。 Pre-denaturation at 94 ℃ for 5 min; each 2 cycle programs: 94 ℃, 1 min, 63 ℃/61 ℃/59 ℃/57 ℃/55 ℃, 1 min, 72 ℃, 1 min; 20 cycle programs: 94 ℃ , 1 min, 55 ℃, 1 min, 72 ℃, 1 min; the final extension at 72 ℃ for 10 min.	Garbeva, et al. 2003^[20]
R1378	CGGTGTGTACAAGGCCCGGGAACG		Garbeva, et al. 2003^[20]
Fusarium
EF-1	ATGGGTAAGGA(A/G)GACAAGAC	94 ℃预变性5 min；30个循环程序：94 ℃，1 min，55 ℃，1 min，72 ℃，1 min；最后72 ℃延伸10 min。 Pre-denaturation at 94 ℃ for 5 min; 30 cycles program: 94 ℃, 1 min, 55 ℃, 1 min, 72 ℃, 1 min; finally extension at 72 ℃ for 10 min.	O’Donnell, et al. 1998^[21]
EF-2	GGA(G/A)GTACCAGT(G/C)ATCATGTT		O’Donnell, et al. 1998^[21]
Alfie-GC	TCGTCATCGGCCACGTCGACTC	94 ℃预变性5 min；35个循环程序：94 ℃，1 min，57 ℃，1 min，72 ℃，50 s；最后72 ℃延伸10 min。 Pre-denaturation at 94 ℃ for 5 min; 35 cycles program: 94 ℃, 1 min, 57 ℃, 1 min, 72 ℃, 50 s; the final extension at 72 ℃ for 10 min.	Yergeau, et al. 2005^[22]
Alfie-2	CCTTACCGAGCTCAGCGGCTTC		Yergeau, et al. 2005^[22]
ITS1F	CTTGGTCATTTAGAGGAAGTAA	94 ℃预变性 5 min；35个循环程序：94 ℃，50 s，60.4 ℃，45 s，72 ℃，60 s；最后72 ℃延伸10 min。 Pre-denaturation at 94 ℃ for 5 min; 35 cycles program: 94 ℃, 50 s, 60.4 ℃, 45 s; the final extension at 72 ℃ for 10 min.	Lievens, et al. 2010^[23]
AFP308R	CGAATTAACGCGAGTCCCAA		Lievens, et al. 2010^[23]
说明：GC夹序列为CGCCCGGGGCGCGCCCCGGGCGGGGCGGGGGCACGGGGGG。 Note: GC clip sequence was CGCCCGGGGCGCGCCCCGGGCGGGGCGGGGGCACGGGGGG.

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表 2 芽孢杆菌属DGGE群落多样性

Table 2. Diversity index based on DGGE of Bacillus

处理 Treatment	辛普森指数（J） Simpson(J)	香农指数（H） Shannon(H)	均匀度 Evenness	布里渊指数 Brillouin
CK	1.0411±0.0019 a	3.9246±0.0074 d	0.9812±0.0019 a	2.7083±0.0656 b
1Y	1.0321±0.0056 b	4.7206±0.0082 b	0.982±0.0017 a	3.1245±0.1575 a
10Y	1.0298±0.0021 b	4.6592±0.0105 c	0.9799±0.0022 a	3.2195±0.0248 a
30Y	1.0242±0.0029 b	4.7973±0.0152 a	0.9777±0.0031 a	3.2214±0.0561 a
注：表中的字母表示同一行数据差异显著（P＜0.05, n=3） Note: Data with different letters indicate significant difference on a same row (P＜0.05, n=3).

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表 3 芽孢杆菌属DGGE条带鉴定

Table 3. Identification of DGGE bands on Bacillus

条带 Bands	CK	1Y	10Y	15Y	同源性相似系数 Homology similarity coefficient/%	物种 Species
B1	0.0719 a	0.0716 a	0.0603 b	0.0670 ab	100	Bacillus sp.芽孢杆菌
B11	0.0000 b	0.0486 a	0.0000 b	0.0476 a	100
B14	0.0371 b	0.0455 a	0.0377 b	0.0366 b	100
B30	0.0223 b	0.0404 a	0.0243 b	0.0243 b	100
B31	0.0000 b	0.0245 a	0.0244 a	0.0208 a	100
B35	0.0498 b	0.0522 b	0.061 a	0.0573 ab	100
B37	0.0000 b	0.0000 b	0.0000 b	0.0352 a	100
B2	0.0758 ab	0.0761 ab	0.0728 b	0.0849 a	100	Bacillus mycoides 蕈状芽孢杆菌
B5	0.0734 a	0.0777 a	0.0754 a	0.0791 a	100	Bacillus mycoides 蕈状芽孢杆菌
B18	0.0000 b	0.0294 a	0.0000 b	0.0000 b	100	Bacillus pumilus 短小芽孢杆菌
B19	0.0000 b	0.0274 a	0.0000 b	0.0000 b	100
B28	0.0285 a	0.0294 a	0.0303 a	0.0293 a	100
B3	0.0777 b	0.0849 ab	0.0770 b	0.0893 a	100	Bacillus stubtilis 枯草芽孢杆菌
B4	0.0755 b	0.0773 ab	0.0746 b	0.0862 a	100
B20	0.0000 c	0.0000 c	0.0603 b	0.0726 a	100
B21	0.0000 c	0.0000 c	0.0653 b	0.0783 a	100
B26	0.0000 c	0.0451 a	0.0406 ab	0.0380 b	100
B27	0.0000 c	0.0000 c	0.0395 a	0.0346 b	100
B6	0.0729 ab	0.0680 b	0.0749 ab	0.0759 a	100	Brachybacterium sp. 短状杆菌
B7	0.0693 b	0.0685 b	0.0768 a	0.0000 c	100	Bacillus cereus 蜡样芽孢杆菌
B8	0.0698 bc	0.0621 c	0.0808 a	0.0751 ab	100
B22	0.0741 b	0.0773 ab	0.0756 b	0.0848 a	100
B24	0.0000 b	0.0445 a	0.0434 a	0.0419 a	100
B25	0.0388 a	0.0462 a	0.0440 a	0.0429 a	100
B12	0.0000 b	0.0237 a	0.0000 b	0.0000 b	100	Stenotrophomonas maltophilia 嗜麦芽糖寡养单胞菌
B17	0.0000 c	0.0305 a	0.0233 b	0.0230 b	100
B34	0.0539 b	0.0602 ab	0.0631 a	0.063 a	100
B13	0.0000 c	0.0411 a	0.0000 c	0.0328 b	100	Bacillus amyloliquefaciens 解淀粉芽孢杆菌
B16	0.0000 b	0.0286 a	0.0000 b	0.0000 b	100
B29	0.0000 b	0.0000 b	0.0248 a	0.0000 b	100
B23	0.0000 b	0.0000 b	0.0549 a	0.0509 a	100	Bacillus odyssey 奥德赛芽孢杆菌
B32	0.0000 b	0.0000 b	0.0000 b	0.0655 a	100	Bacillus halodurans 耐盐芽孢杆菌
B33	0.0000 b	0.0000 b	0.0000 b	0.0643 a	100	Bacillus halodurans 耐盐芽孢杆菌
B9	0.0618 a	0.0496 b	0.0527 b	0.0580 ab	100	Uncultured bacterium未知菌种
B10	0.0000 b	0.0493 a	0.0460 a	0.0475 a	100
B15	0.0000 b	0.0372 a	0.0000 b	0.0000 b	100
B36	0.0000 c	0.0456 a	0.0369 b	0.0363 b	100
注：表中数据为各条带灰度值（通过条带的面积与亮度进行计算）。数据后不同小写字母表示差异显著（P ＜0.05）。表5同。 Note: Data are gray values calculated with area and brightness of individual bands; those with different lowercase letters indicate significant difference at P＜0.05 level. Same for Table 5.

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表 4 镰刀菌属DGGE群落多样性

Table 4. Diversity index based on DGGE of Fusarium

处理 Treatment	辛普森指数（J） Simpson(J)	香农指数（H） Shannon(H)	均匀度 Evenness	布里渊指数 Brillouin
CK	0.8783±0.0043 a	3.1458±0.0164 a	0.9093±0.0048 b	2.8925±0.0136 a
1Y	0.7666±0.0029 d	2.2739±0.0194 d	0.8797±0.0075 c	2.1294±0.0211 d
10Y	0.8064±0.0038 c	2.4497±0.0126 c	0.9477±0.0049 a	2.3049±0.0232 c
15Y	0.8580±0.0017 b	2.897±0.0144 b	0.9139±0.0046 b	2.6865±0.0086 b

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表 5 镰刀菌属DGGE条带鉴定

Table 5. Identification of DGGE bands on Fusarium

条带 Bands	CK	1Y	10Y	15Y	同源性相似系数 Homology similarity coefficient/%	物种 Species
F1	0.0385 b	0.0000 c	0.1395 a	0.0000 c	100	Fusarium solani 腐皮镰刀菌
F3	0.0277 b	0.0000 c	0.0000 c	0.0407 a	100
F4	0.0122 a	0.0000 b	0.0000 b	0.0000 b	100
F9	0.0000 b	0.0549 a	0.0000 b	0.0000 b	100
F10	0.0000 b	0.0614 a	0.0000 b	0.0000 b	100
F11	0.0000 d	0.2643 a	0.1374 b	0.1148 c	100
F13	0.1642 a	0.1215 b	0.0986 b	0.0236 c	100
F2	0.0500 b	0.0000 c	0.1058 a	0.0000 c	100	Fusarium oxysporum 尖孢镰刀菌
F6	0.1017 b	0.0000 c	0.0000 c	0.1582 a	100
F8	0.0814 a	0.0000 c	0.0000 c	0.0509 b	100
F12	0.1384 b	0.1360 b	0.1978 a	0.1364 b	100
F5	0.0828 a	0.0000 b	0.0000 b	0.0000 b	100	Fusarium equiseti 木贼镰刀菌
F7	0.0864 b	0.0000 c	0.0000 c	0.1975 a	100	Fusarium sp. 镰刀菌
F14	0.0000 b	0.0000 b	0.1197 a	0.0000 b	100	Fusarium sp. 镰刀菌
F15	0.0000 b	0.0000 b	0.0000 b	0.0645 a	100	Aspergillus nidulans 小巢状曲菌

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