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2019 , Vol. 40 >Issue 9: 1847 - 1857

DOI: https://doi.org/10.3969/j.issn.1000-2561.2019.09.025

Ecology

The Autotoxicity of Tea Tree Rhizosphere Soil Chemicals and the Effect of Soil Microbial Diversity

  • WANG Haibin 1, 2 ,
  • CHEN Xiaoting 1, 2 ,
  • ZHAO Hu 1, 3 ,
  • WANG Yuhua 1 ,
  • ZHANG Qingxu 2 ,
  • WANG Peng 2 ,
  • YE Jianghua 2, 4 ,
  • DING Li 1
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  • 1. College of Life Sciences, Longyan University, Longyan, Fujian 364012, China
  • 2. Fujian Provincial Key Laboratory of Agroecological Processing and Safety Monitoring, Fujian Agriculture and Forestry University, Fuzhou, Fujian 350002, China
  • 3. College of Horticulture, Gansu Agricultural University, Lanzhou, Gansu 730070, China
  • 4. College of Tea and Food Science, Wuyi University, Wuyishan, Fujian 354300, China

Received date: 2019-02-26

  Request revised date: 2019-04-10

  Online published: 2019-09-10

Copyright

Copyright reserved © 2019. Office of Acta Agronomica Sinica All articles published represent the opinions of the authors, and do not reflect the official policy of the Chinese Medical Association or the Editorial Board, unless this is clearly specified.
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Abstract

In order to analyze the effect of tea tree rhizosphere soil chemicals on the soil microbial diversity, the rhizosphere soils from Tieguanyin tea plantations of 0, 3, 9 and 25 years old were extracted and eluted through different polar resins to discuss the autotoxicity of resins eluted and the effect of soil microbial diversity. The results showed that the inhibitory effect of ADS-7 resin eluent on the root length of receptor was the strongest. After the replanted tea trees was treated by ADS-7 resin eluent, the number of bacteria in the tea trees rhizosphere soil decreased with the increase of planting soil age. Correlation analysis result showed that 15 T-RFs from bacterial community were significantly and positively correlated with soil age, which included 31 species of microbes belonging to 8 classes, respectively. Thirty-one microbes could be divided into 4 types according to the function, among them, 19 species of pathogenic bacteria accounted for 61.29%. In addition, 18 T-RFs were significantly and negatively correlated with soil age, which included 31 species of bacteria belonging to 11 classes, respectively. The thirty-one bacteria could be divided into 6 types according to the function, and total percentage of the bacteria to inhibit 83.87%. In brief, after the replanted tea trees was treated by ADS-7 resin eluent, the number of pathogenic bacteria in tea tree pathogenic bacteria, carbon cycle, nitrogen cycle, sulphur cycle and the bacteria to improve the soil quality accounted for rhizosphere soil was great enhanced and the number of probiotics and soil nutrient cycling bacteria decreased significantly as planting soil age increased, which led to the imbalance of soil microbial ecosystem.

Key words： rhizosphere soil of tea tree; autotoxicity; chemical; microbial diversity

Cite this article

WANG Haibin , CHEN Xiaoting , ZHAO Hu , WANG Yuhua , ZHANG Qingxu , WANG Peng , YE Jianghua , DING Li . The Autotoxicity of Tea Tree Rhizosphere Soil Chemicals and the Effect of Soil Microbial Diversity[J]. Chinese Journal of Tropical Crops, 2019 , 40(9) : 1847 -1857 . DOI: 10.3969/j.issn.1000-2561.2019.09.025

茶树[Camellia sinensis (L.) O. Kuntze]属于山茶科、山茶属灌木或小乔木茶种,为多年生常绿木本植物。铁观音茶园的开垦、种植到采摘需要2~3年,正常经济效益旺期在7年左右。21世纪初,安溪铁观音进入飞速发展时期。大型茶叶企业纷纷在安溪县及周边县市大量开垦新茶园,种植铁观音茶树,并按公司化方式统一管理,安溪县周围的山地已形成以茶树为主要植物种群的单一群落结构。为此,茶园“土壤病”形成,茶园逐渐退化,茶树病虫害加剧,茶叶单产水平及品质也逐年下降。茶园退化一方面是茶树本身自然衰老,另一方面茶树连年种植后,土壤环境发生变化,不利于茶树生长的因素积累,土壤自毒作用加剧。
王海斌等[1]调查分析了安溪县9个乡镇茶园土壤的酸化情况发现,调查的茶园中37.67%的土壤已经酸化,10.03%的土壤不适宜种植茶树,茶树树龄与其根际土壤pH呈极显著负相关,茶叶的产量、品质与茶树根际土壤pH呈极显著正相关,该研究认为,茶树根际土壤酸度随着茶树树龄的增加而加剧,茶叶产量降低、品质呈现下降趋势。Ye等[2,3]研究也发现,随着茶树树龄的增加,茶树根际土壤酸度加剧,自毒作用潜力增强,茶叶产量和品质降低,这种现象的形成与土壤中酸类物质的积累增加有关。本课题组前期对不同树龄茶树根际土壤物质进行HPLC分析发现,随着茶树树龄的增加,茶树根际土壤中6种酸类物质含量不断上升。此外,进一步采用不同极性树脂吸附土壤物质并进行生物测试发现,以ADS-7树脂吸附后的洗脱液自毒作用能力最强,GC-MS分析ADS-7树脂洗脱液的物质成分,发现13种物质随着茶树树龄的增加呈现上升趋势,其中9种是酸类物质[4,5]。可见,退化茶园“土壤病”的形成与茶树根际土壤物质种类与数量密切相关。
土壤是一个复杂生态系统,存在着丰富的微生物种类,植物释放和积累的物质通过土壤载体进行传播,进而影响土壤中的微生物区系与种类,土壤生态系统朝着专一化的趋势发展[6,7,8]。综上可见,随着茶树树龄的增加,土壤自毒潜力加剧,茶树根际土壤微生物发生显著变化[9,10];而关于这种变化是否与土壤物质有关,土壤物质对微生物多样性变化是否会产生影响的研究还鲜有报道。据此,本研究以不同树龄铁观音茶树根际土壤为材料,采用不同极性树脂进行吸附并洗脱,土壤洗脱液一方面用于自毒潜力评价,一方面用于处理新种植的茶树并测定茶树根际土壤微生物种类、群落结构及其功能的变化,以期为茶园土壤退化的修复提供一定的理论依据。

1 材料与方法

1.1 材料

以福建省泉州市安溪县龙涓乡铁观音茶园为研究地点（东经117°93′、北纬24°97′）,收集已种植0、3、9、25年的铁观音茶树根际土壤,用于土壤物质提取及不同极性树脂的吸附与洗脱后的生物测试。土壤取样方法：随机选择3、9、25 a树龄的茶树各100株,去除土壤表层杂质,连根挖出茶树,收集茶树根际土壤;以未种植过茶树的土壤为对照（0 a）,首先去除地表植被和杂质,收集15~25 cm范围的土壤,多点随机收集;各样品的取样量均为约15 kg。取样点茶园土壤的基本理化指标：有机质、全氮、全磷、全钾、速效氮、速效磷、速效钾含量,分别为8.34 g/kg、2.17 g/kg、1.05 g/kg、1.46 g/kg,25.3 mg/kg、56.7 mg/kg、264.6 mg/kg。

1.2 方法

1.2.1 不同极性树脂洗脱液对受体莴苣（Lactuca saliva）根长的抑制率分析 土壤样品置于阴凉处自然风干,研磨过40目筛,称取0、3、9、25 a茶树根际土壤各5 kg,加入20 L蒸馏水,360 W超声提取1 h（每隔10 min均匀搅拌1次）,120 r/min振荡1 h,重复5次,过滤,提取液于45 ℃下旋转蒸发浓缩至5 L;此时,每毫升浸提液约含有1 g土壤物质[11]。
先将不同极性的树脂ADS-7、ADS-21、ADS-F8、ADS-17、ADS-8（天津南开合成科技有限公司）采用纯乙醇浸泡活化24 h,蒸馏水浸泡清洗至没有乙醇。取收集的不同样品提取浓缩液各5 L,分成5组,每组1 L,采用5种不同极性树脂分别进行静态吸附,方法为：每升提取浓缩液加入200 g树脂,置于摇床上120 r/min振荡吸附24 h,弃上清液,树脂中加入600 mL甲醇,置于摇床上120 r/min洗脱12 h,收集甲醇洗脱液过滤并浓缩至200 mL,用于不同极性树脂洗脱液对受体莴苣根长的抑制率测定,具体参照Wang等[12]的方法进行测定,根长抑制率的计算公式为,相对抑制率=(1-处理值/对照值)×100%。
1.2.2 外源添加不同极性树脂洗脱液处理新种植的茶树 取不同极性树脂吸附后的洗脱液各50 mL,于45 ℃下旋转蒸发浓缩至10 mL,加无菌蒸馏水定容至2 L,-20 ℃保存备用。
取未种植过茶树的土壤风干研磨并过40目筛;将土壤装入盆中,每盆10 kg,选择1年生、长势相对一致的铁观音茶苗并移栽到盆中,每盆6株,移栽后,恢复生长30 d。适当搅动、松动种植土壤,将配置好的树脂洗脱液2 L缓慢倒入盆中,尽量使其在土壤中分布均匀,继续常规种植茶树60 d,收集茶树根际土壤,用于土壤微生物的T-RFLP分析,每种处理种植3盆,即3个重复。种植土壤的基本理化指标为：有机质、全氮、全磷、全钾、速效氮、速效磷、速效钾含量,分别为9.02 g/kg、1.03 g/kg、0.56 g/kg、1.85 g/kg、89.46 mg/kg、15.28 mg/kg、179.62 mg/kg。
1.2.3 土壤微生物的T-RFLP分析 采用CTAB-蛋白酶K-液氮冻融法直接抽提不同样品的土壤微生物总DNA,用于微生物的16S rDNA的扩增和酶切[13]。16S rDNA扩增的PCR反应程序为：94 ℃ 5 min;94 ℃ 45 s,52 ℃ 45 s,72 ℃ 1.5 min,30个循环后,72 ℃ 10 min。其中,扩增引物采用带有FAM荧光标记的细菌通用引物,分别为8F-FAM（5′-AGAGTTTGATCCTGGCTCAG-3′）和926R（5′-CCGTCAATTCCTTTRAGTTT-3′）,PCR反应体系的总体积为25 μL,反应体系中各成分含量为：2.5 μL 10×PCR Buffer、正反向引物（10 μmol/L）各0.8 μL、2.0 μL dNTP（25 μmol/L）、0.2 μL BSA、17.05 μL ddH2O、0.15 μL rTaq、1.5 μL DNA模板。PCR结束后,电泳检测,UNIQ-10柱式DNA胶回收试剂盒回收PCR产物中1000 bp左右的片段,用于酶切。
酶切采用HaeIII和MspI 2种内切酶进行消化,其中HaeIII酶切体系为：HaeIII 1 μL、H×Buffer 2 μL、ddH2O 7 μL、PCR产物10 μL;MspI酶切体系为：MspI 1 μL、T-Buffer 2 μL、BSA 2 μL、ddH2O 5 μL、PCR产物10 μL。将配置好的2种酶切体系分别置于37 ℃水浴酶切5 h,酶切后的产物采用ABI自动测序分析仪（Model 3130 Applied Biosystems）测定。

1.3 数据处理

土壤微生物测序结果分析采用GeneMarker V1.2软件,分析参数参照SoftGenetics Application Note July, 2006。酶切获得的T-RFs片段分析采用Ribosomal Database Project II数据库比对法,获取T-RFs双酶切片段所对应的微生物物种。T-RFs片段丰度计算公式为：T-RFs片段丰度=ni/N×100,式中ni代表可分辨的T-RF的峰面积,N代表所有T-RF峰面积的总和[14]。其余常规的数据分析、方差分析、显著性分析及变化分析等采用Excel软件和DPS数据处理系统进行处理。

2 结果与分析

2.1 不同极性树脂洗脱液对受体莴苣根长的抑制率分析

不同极性树脂洗脱液对受体莴苣根长影响的分析结果表明（表1）,不同极性树脂洗脱液对受体根长均存在一定的抑制作用,以25 a茶树根际土壤的树脂吸附洗脱液抑制作用最强,表现为ADS-8、ADS-17、ADS-F8、ADS-21、ADS-7树脂洗脱液对莴苣根长的抑制率分别为15.76%、19.68%、20.57%、29.07%、43.76%。其中以25 a茶树根际土壤经ADS-7树脂吸附的洗脱液抑制作用最强。
表1 不同极性树脂洗脱液对莴苣根长的抑制率

Tab. 1 Inhibition rate of lettuce root length by different polarity resin eluent %

土壤Soil ADS-8 ADS-17 ADS-F8 ADS-21 ADS-7
0 a 0.58±0.25d 2.37±0.35d 0.80±0.32d 1.29±0.36d 2.83±0.34d
3 a 1.82±0.36c 7.56±0.46c 3.24±0.47c 4.54±0.49c 8.50±0.48c
9 a 4.35±0.28b 16.31±0.63b 16.69±0.51b 14.24±0.61b 21.20±0.56b
25 a 15.76±0.62a 19.68±0.39a 20.57±0.72a 29.07±0.66a 43.76±0.71a

Note: Different lowercase letters in the same column indicate significant differences (P<0.05).

注：同列不同小写表示差异显著（P<0.05）。

2.2 ADS-7树脂洗脱液处理后茶树根际土壤细菌多样性分析

由图1可见,0、3、9、25 a茶树根际土壤经ADS-7树脂洗脱液处理后,根际土壤细菌
HaeIII和MspI酶切产物的T-RFs片段,随着土壤年限的增加呈现下降趋势,表现为HaeIII酶切产物的T-RFs片段数量从106个下降至58个,而MspI酶切产物的T-RFs片段数量则从124个下降至68个。可见,不同种植年限土壤的ADS-7树脂吸附洗脱液处理后,茶树根际土壤细菌多样性发生显著的变化。
图1 不同年限土壤的ADS-7树脂吸附洗脱液处理后茶树根际土壤细菌MspI和HaeIII酶切产物的T-RFs片段

不同小写字母表示不同年限土壤T-RFs差异显著（P<0.05）。

Fig. 1 The T-RFs numbers digested by MspI and HaeIII of tea tree rhizospheric soils bacteria after treated by different soil’s ADS-7 resin adsorption eluent

Different lowercase letters indicate significant differences (P<0.05).

2.3 ADS-7树脂洗脱液处理后茶树根际土壤正、负相关细菌分析

以不同茶树根际土壤细菌HaeIII酶切后的T-RFs片段丰度进行相关性分析发现（图2）,不同年限茶树根际土壤的ADS-7树脂吸附洗脱液处理后,与土壤种植年限呈显著或极显著正相关的细菌T-RFs片段共15个,呈显著或极显著负相关的有18个。
图2 不同年限土壤的ADS-7树脂吸附洗脱液处理后茶树根际土壤正、负相关细菌T-RFs丰度

A：正相关微生物系统分类;B：负相关微生物系统分类。

Fig. 2 The positively and negatively correlated bacteria T-RFs abundances of tea tree rhizospheric soil after treated by different soil’s ADS-7 resin adsorption eluent

A: Positive correlate microbial communities; B: Negative correlate microbial communities.

进一步将T-RFs片段与Ribosomal Database Project II数据库进行比对,结果表明（图3）,正相关细菌T-RFs片段15个,涉及31种细菌,由8个纲组成,占比分别为β-变形菌纲12.90%、γ-变形菌纲16.13%、δ-变形菌纲6.45%、芽孢杆菌纲38.71%、放线菌亚纲9.68%、鞘脂杆菌纲9.68%、梭菌纲3.23%、螺旋体纲3.23%;负相关细菌T-RFs片段18个,涉及31种细菌,由11个纲组成,占比分别为α-变形菌纲25.81%、β-变形菌纲6.45%、γ-变形菌纲12.90%、放线菌亚纲19.35%、梭菌纲12.90%、芽孢杆菌纲6.45%、螺旋体纲3.23%、柔膜菌纲3.23%、鞘脂杆菌纲3.23%、红色杆菌亚纲3.23%、梭杆菌纲3.23%。可见,不同种植年限土壤的ADS-7树脂吸附洗脱液处理后,茶树根际土壤细菌种类发生显著变化。
图3 不同年限土壤的ADS-7树脂吸附洗脱液处理后茶树根际土壤正、负相关细菌的系统分类

A：正相关微生物系统分类;B：负相关微生物系统分类。

Fig. 3 The positively and negatively correlated bacteria system classification of tea tree rhizospheric soil after treated by different soil’s ADS-7 resin adsorption eluent

A: Positive correlate microbial communities; B: Negative correlate microbial communities.

2.4 ADS-7树脂洗脱液处理后茶树根际土壤正相关细菌功能分析

由表2可见,0、3、9、25年茶树根际土壤经ADS-7树脂洗脱液处理后,茶树根际土壤正相关细菌有31种,按照其功能可分为4大类：病原菌19种,占比61.29%;抑制病原菌相关细菌3种,占比9.68%;碳素循环相关细菌4种,占比12.90%;改善土壤质地相关细菌5种,占比16.13%。可见,不同种植年限土壤的ADS-7树脂吸附洗脱液处理后,茶树根际土壤不同功能类别的细菌数量发生显著变化,特别是病原菌数量显著增加,表现为正相关细菌中病原菌占比最大。
表2 不同年限土壤经ADS-7树脂吸附洗脱液处理后茶树根际土壤正相关细菌类群及功能

Tab. 2 The positively correlated bacteria flora and its function in rhizospheric soils after treated by different soil’s ADS-7 resin adsorption eluent

功能 Function haeⅢ mspⅠ 种名 Species[a] 土壤细菌相对丰度
The relative abundance of bacteria community/%		 相关系数
Correlation coefficient
0 a 3 a 9 a 25 a
病原菌
Pathogenic bacteria		 39 496 弗氏柠檬酸杆菌 Citrobacter freundii、克氏柠檬酸杆菌 Citrobacter koseri、耐酸木杆菌 Xylella fastidiosa、黄瓜欧文氏菌 Erwinia tracheiphila、柠檬酸杆菌 Citrobacter、布氏柠檬酸杆菌 Citrobacter braakii[15,16,17,18,19] 0.3 0.5 0.8 1.4 0.99**
209 155 巴氏葡萄球菌 Staphylococcus pasteuri[20] 0.1 0.1 0.3 0.4 0.93*
216 73 产气真杆菌 Eubacterium aerofaciens[21] 0.2 0.3 0.5 0.6 0.92*
218 140 多形链球菌 Streptococcus pleomorphus[22] 0.1 0.2 0.6 0.7 0.88*
229 280 节杆菌 Arthrobacter、驹形白色杆菌 Leucobacter komagatae[23,24] 0.2 0.4 0.5 0.8 0.96**
230 140 霍氏厌氧分支杆菌 Anaerobranca horikoshii[25] 0.4 0.5 0.5 0.8 0.98**
310 155 表皮葡萄球菌 Staphylococcus epidermidis、琥珀葡萄球菌 Staphylococcus succinus、山羊葡萄球菌 Staphylococcus caprae、马胃葡萄球菌 Staphylococcus equorum、路邓葡萄球菌 Staphylococcus lugdunensis、好气金黄色葡萄球菌 Staphylococcus aerophilus、金黄色葡萄球菌 Staphylococcus aureus subsp.、头状葡萄球菌 Staphylococcus capitis subsp[26,27] 0.2 0.3 0.7 1.2 0.99**
抑制病原菌
The bacteria inhibit pathogenic bacteria		 223 139 土壤芽胞杆菌 Bacillus edaphicus[28] 0.3 0.5 0.6 0.9 0.97**
225 496 粪产碱菌粪亚种 Alcaligenes faecalis subsp.[28] 0.1 0.3 0.3 0.5 0.97**
226 139 强壮类芽胞杆菌 Paenibacillus validus[28] 0.4 0.7 0.9 1.3 0.96**
碳素循环
Carbon cycle		 39 91 发酵噬纤维菌 Cytophaga fermentans[29] 0.3 0.5 0.8 1.4 0.99**
213 482 嗜甲基菌 Methylophilus methylotrophus[30] 0.2 0.5 0.7 0.8 0.88*
225 496 粪透明颤菌 Vitreoscilla stercoraria[31] 0.1 0.3 0.4 0.7 0.97**
282 91 溶解噬纤维菌 Cytophaga lytica[29] 0.4 0.6 0.7 1.2 0.99**
改善土壤质地
Improve soil texture		 39 142 食醇鞘氨醇杆菌 Sphingobacterium spiritivorum[32] 0.3 0.5 0.8 1.4 0.99**
215 80 异化金属还原菌 Geobacter metallireducens[33] 0.2 0.3 0.6 0.8 0.95*
229 280 酯香微杆菌 Microbacterium esteraromaticum[34] 0.3 0.5 0.8 1.1 0.96**
256 280 橙黄螺旋体 Spirochaeta aurantia subsp. Aurantia[35] 0.2 0.2 0.4 0.7 0.99**
295 152 鞘氨醇单胞菌 Sphingomonas Lep1[32] 0.1 0.3 0.6 0.7 0.88*

Note: * indicates significant correlation at 0.05 level; ** indicates extremely significant correlation at 0.01 level; [a] the reference of microbial function.

注：*表示显著相关（P<0.05）;**表示极显著相关（P<0.01）;[a]微生物功能文献。

2.5 ADS-7树脂洗脱液处理后茶树根际土壤负相关细菌功能分析

由表3可见,0、3、9、25 a茶树根际土壤经ADS-7树脂洗脱液处理后,茶树根际土壤负相关细菌31种,按照其功能可分为6大类：病原菌5种,占比16.13%;抑制病原菌相关细菌4种,占比12.90%;碳素循环相关细菌10种,占比32.26%;氮素循环相关细菌8种,占比25.81%;改善土壤质地相关细菌3种,占比9.68%;硫素循环细菌1种,占比3.23%。可见,不同种植年
限土壤经ADS-7树脂吸附洗脱液处理后,茶树根际土壤不同功能类别的细菌数量发生显著变化,特别是与抑制病原菌、碳素循环、氮素循环、土壤质地改善、硫素循环相关的细菌数量显著下降,总占比达到83.87%。
表3 不同年限土壤经ADS-7树脂吸附洗脱液处理后茶树根际土壤负相关细菌类群及功能

Tab. 3 The negatively correlated bacteria flora and its function in rhizospheric soils after treated by different soil’s ADS-7 resin adsorption eluent

功能 Function haeⅢ mspⅠ 种名 Species[a] 土壤细菌相对丰度
The relative abundance of bacteria community/%		 相关系数
Correlation coefficient
0 a 3 a 9 a 25 a
病原菌
Pathogenic bacteria		 67 160 赫氏蜱疏螺旋体 Borrelia hermsii[36] 0.7 0.6 0.4 0.2 -0.97**
194 152 克利夫兰阿菲波菌 Afipia clevelandensis[37] 0.8 0.6 0.4 0.3 -0.88*
237 152 死亡梭杆菌 Fusobacterium mortiferum[38] 1.1 0.8 0.6 0.5 -0.88*
250 73 非凡螺原体 Spiroplasma mirum[39] 0.5 0.5 0.2 0.1 -0.90*
273 152 亚洲分枝杆菌 Mycobacterium asiaticum[40] 0.8 0.7 0.4 0.2 -0.95*
续表3 不同年限土壤经ADS-7树脂吸附洗脱液处理后茶树根际土壤负相关细菌类群及功能

Tab. 3 The negatively correlated bacteria flora and its function in rhizospheric soils after treated by different soil’s ADS-7 resin adsorption eluent (continued)

功能 Function haeⅢ mspⅠ 种名 Species[a] 土壤细菌相对丰度
The relative abundance of bacteria community/%		 相关系数
Correlation coefficient
0 a 3 a 9 a 25 a
抑制病原菌The bacteria inhibit pathogenic bacteria 67 160 草分枝杆菌 Mycobacterium phlei[41] 0.7 0.6 0.4 0.2 -0.97**
231 93 产碱杆菌 Alcaligenes[28] 1.3 1.2 0.7 0.3 -0.96**
237 152 马阔里类芽胞杆菌 Paenibacillus macquariensis[42] 1.1 0.8 0.6 0.5 -0.88*
253 490 虱沃尔巴克氏体 Wolbachia persica[43] 1.0 0.8 0.5 0.1 -0.98**
碳素循环
Carbon cycle		 67 279 耻垢分枝杆菌 Mycobacterium smegmatis、抗热分枝杆菌 Mycobacterium thermoresistibile[44] 0.7 0.6 0.4 0.2 -0.97**
69 139 液泡外硫红螺菌 Ectothiorhodospira vacuolata、沙氏外硫红螺菌 Ectothiorhodospira shaposhnikovii[45] 1.2 0.8 0.5 0.1 -0.94*
195 152 沼泽红假单胞菌 Rhodopseudomonas palustris[44] 1.4 0.9 0.6 0.2 -0.92*
200 490 产甲酸草酸杆菌 Oxalobacter formigenes[46] 0.5 0.3 0.3 0.1 -0.92*
239 432 蜂房类芽胞杆菌 Paenibacillus alvei[47] 0.9 0.7 0.4 0.3 -0.88*
243 455 球形红球形菌 Rhodopila globiformis[48] 1.2 1.0 0.7 0.4 -0.96**
245 146 产色高温单孢菌 Thermomonospora chromogena[49] 0.8 0.6 0.3 0.2 -0.88*
253 425 甲烷甲基单胞菌 Methylomonas methanica[50] 1.0 0.8 0.5 0.1 -0.98**
氮素循环
Nitrogen cycle		 195 152 玫瑰色红游动菌 Rhodoplanes roseus、维氏硝化杆菌 Nitrobacter winogradskyi、大豆慢生根瘤菌 Bradyrhizobium japonicum[51,52,53] 1.4 0.9 0.6 0.2 -0.92*
210 138 耐辐照红色杆菌 Rubrobacter radiotolerans[54] 1.1 0.7 0.5 0.2 -0.91*
212 160 嗜盐梭菌 Clostridium halophilum[55] 0.9 0.7 0.3 0.1 -0.92*
224 400 苜蓿中华根瘤菌 Sinorhizobium meliloti[56] 0.7 0.4 0.2 0.1 -0.88*
243 180 乙酰微小杆菌 Exiguobacterium acetylicum[57] 1.2 1.0 0.7 0.4 -0.96**
285 91 成团微颤蓝细菌 Microscilla aggregans[58] 1.1 0.8 0.5 0.3 -0.90*
改善土壤质地
Improving soil texture		 67 142 简单类诺卡氏菌 Nocardioides simplex[59] 0.7 0.6 0.4 0.2 -0.97**
195 152 扭脱甲基杆菌 Methylobacterium extorquens[60] 1.4 0.9 0.6 0.2 -0.92*
212 138 脱卤脱亚硫酸菌 Desulfitobacterium dehalogenans[61] 0.9 0.7 0.3 0.1 -0.92*
硫素循环
Sulphur cycle		 258 282 生黄瘤胃球菌 Ruminococcus flavefaciens[62] 0.7 0.6 0.4 0.3 -0.92*

Note: * indicates significant correlation at 0.05 level; ** indicates extremely significant correlation at 0.01 level; [a] the reference of microbial function.

注：*表示显著相关（P<0.05）;**表示极显著相关（P<0.01）;[a]微生物功能文献。

3 讨论

植物土壤生态系统,主要涉及植物、土壤、微生物三者,三者之间相互协调,影响着植物的生长,土壤的质量,微生物的生存[63]。茶树长期种植后,根系分泌物在土壤中大量积累,使土壤微生物在选择性压力影响下发生了显著的变化,这种变化可能朝着对茶树生长有利或有弊的方向发展。本研究结果表明,随着茶树树龄的增加,茶树根际土壤不同极性树脂吸附洗脱液对受体莴苣根长存在一定的抑制作用,以ADS-7树脂洗脱液的抑制作用最强。其次,不同种植年限土壤的ADS-7树脂吸附洗脱液处理重新种植的茶树后发现,随着土壤种植年限的增加,茶树根际土壤细菌数量呈现下降趋势。众多学者在研究不同作物——太子参、山银花、茶树等连续种植后,土壤微生物数量变化时也发现类似的趋势[64,65,66]。可见,ADS-7树脂洗脱液处理对茶树根际土壤微生物数量产生影响,这种影响与不同年限茶树原位种植现象类似。
作物长期种植,土壤物质积累后会对微生物数量与种类产生影响。Wang等[12]研究发现,百香果连续种植后,产量和品质呈现下降趋势;郝慧荣等[67]研究发现,牛膝连年种植后,反而有利于促进其生长和品质提高。可见,深入分析作物连续种植后土壤微生物种类及功能变化对于明晰“土壤病”的形成具有重要的意义。本研究结果表明,不同种植年限土壤的ADS-7树脂吸附洗脱液处理重新种植的茶树后,随着土壤年限的增加,与其正相关细菌T-RFs片段15个,涉及8个纲、31种细菌,其中病原菌19种,占比61.29%。可见,ADS-7树脂吸附洗脱液处理后,茶树根际土壤的病原菌数量增多,进而影响茶树的生长。此外,进一步分析发现,随着土壤年限的增加,与其负相关细菌T-RFs片段18个,涉及11个纲、31种细菌,其中与抑制病原菌、碳素循环、氮素循环、土壤质地改善、硫素循环相关的细菌数量显著下降,总占比达到83.87%。可见,ADS-7树脂吸附洗脱液处理后,茶树根际土壤的益生菌与土壤养分循环相关的细菌数量下降,茶树根际土壤质地变劣,养分循环受阻。
土壤微生物是土壤生态系统的重要组成部分,其数量及多样性水平高低对于生态系统稳定具有重要的作用,丰富的土壤微生物有利于降低“土壤病”的发生机率,反之则提高[68,69]。可见,不同年限土壤的ADS-7树脂吸附洗脱液处理重新种植的茶树后,茶树根际土壤细菌多样性及群落结构失去平衡,进而可能导致茶树生长受阻。
综上,本研究探讨了茶树根际土壤物质的自毒潜力及其对土壤微生物多样性的影响,结果表明,ADS-7树脂吸附洗脱液对受体莴苣根长的抑制作用最强,ADS-7树脂洗脱液处理重新种植的茶树后,随着土壤年限的增加,茶树根际土壤病原菌数量大幅上升,益生菌与土壤养分循环相关的细菌数量显著下降,土壤微生物生态系统平衡失调,“土壤病”形成,进而可能导致茶树生长和品质受阻。然而,对于茶树—土壤—微生物,三者之间是如何实现相互调控与影响,还需进一步深入研究。

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