生物炭对三七连作土壤性质及真菌群落的影响

doi:10.13560/j.cnki.biotech.bull.1985.2022-0583

摘要/Abstract

摘要：

研究生物炭对三七根际土壤理化性质及真菌群落结构的影响，有助于深入了解三七连作土壤状况，为土壤改良及三七生态种植提供科学依据和理论指导。该研究选取烟杆炭TB，稻壳炭RB为试验材料，以不添加生物炭处理为对照CK，采用田间试验，通过高通量测序技术进行分析，研究两种生物炭对两年生三七根际土壤理化性质及真菌群落结构的影响。施加生物炭能提高三七的存活率，pH值、土壤有效磷、微生物量碳及有效钾对三七成活率具有促进作用，铵态氮对三七成活率具有抑制作用，土壤pH值对酸性磷酸酶和脲酶活性具有正贡献，而对蔗糖酶具有抑制作用。通过对真菌群落结构多样性分析，施加两种生物炭均显著改善了三七连作地的真菌群落结构，提高三七根际土壤真菌的多样性及丰富度指数。不同处理下土壤真菌群落结构存在明显分化，施加生物炭后均显著提升Ascomycota（子囊菌门）的相对丰度，显著降低Basidiomycota（担子菌门）及Mortierellomycota（被孢菌门）的相对丰度，在属水平上，与CK相比，施加生物炭后Fusarium（镰刀菌属）、Aspergillus（曲霉菌属）和Alternaria（链格孢属）的相对丰度显著降低（P<0.05），Botryotinia的丰度显著升高（P<0.01）。冗余分析表明，pH值、硝态氮、有效磷及有效钾是造成三七真菌群落结构与多样性差异的主要环境因子。生物炭通过提高土壤pH值、有效磷、微生物量碳等含量及真菌群落多样性，降低病原菌丰度，从而提高三七成活率。

关键词: 三七, 连作障碍, 生物炭, 根际真菌群落, 高通量测序

Abstract:

Study the effects of biochar on the physical and chemical properties of Panax notoginseng rhizosphere soil and fungal community structure will be conducive to deeply understanding the soil condition of continuous cropping of P. notoginseng, and to providing scientific basis and theoretical guidance for soil improvement and ecological planting of P. notoginseng. In this study, tobacco stalk carbon TB, rice husk carbon RB were selected as experimental materials, and the treatment without biochar as control CK. A field experiment was conducted to study the effects of two kinds of biochar on the physical and chemical properties of biennial P. notoginseng rhizosphere soil and the structure of fungal community. Analysis was performed by high-throughput sequencing technology. The results showed that the application of biochar improved the survival rate of P. notoginseng, and the pH value, soil available phosphorus, microbial biomass carbon and available potassium promoted the survival rate of P. notoginseng, while ammonium nitrogen inhibited the survival rate of P. notoginseng. Soil pH value presented a positive contribution to acid phosphatase and urease activities, but had an inhibitory effect on invertase. Through the analysis of fungal community diversity, adding two biochar significantly improved the fungal community structure in P. notoginseng continuous cropping land, and improved the diversity and richness of soil fungal in the rhizosphere of P. notoginseng. There was obvious differentiation in the community structures under different treatments. After applying biochar, the relative abundance of Ascomycota significantly increased, and the relative abundance of Basidiomycota and Mortierellomycota significantly reduced. At the genus level, compared with the CK, the relative abundance of Fusarium, Aspergillus and Alternaria decreased significantly（P< 0.05）. The abundance of Botryotinia increased significantly（P< 0.01）. Combined with Pearson correlation analysis and RDA, pH value, nitrate nitrogen, available phosphorus and available potassium were the main environmental factors causing the community structure and diversity of P. notoginseng fungi. Biochar results in the survival rate increasing of P. notoginseng by increasing soil pH value, available phosphorus, microbial biomass carbon content and fungal community diversity and reducing the abundance of pathogenic bacteria.

Key words: Panax notoginseng, obstacle in continuous cropping, biochar, rhizosphere fungal community, high-throughput sequencing

孙海航, 官会林, 王旭, 王童, 李泓霖, 彭文洁, 刘柏桢, 樊芳玲. 生物炭对三七连作土壤性质及真菌群落的影响[J]. 生物技术通报, 2023, 39(2): 221-231.

SUN Hai-hang, GUAN Hui-lin, WANG Xu, WANG Tong, LI Hong-lin, PENG Wen-jie, LIU Bo-zhen, FAN Fang-ling. Effects of Biochar on the Soil Properties and Fungal Community Structure under Continuous Cropping of Panax notoginseng[J]. Biotechnology Bulletin, 2023, 39(2): 221-231.

图/表 8

表1 生物炭基本性质

Table 1 Basic properties of the biochar

处理Treatment	pH	电导率 EC/（ms·cm^-1）	铵态氮 NH₄⁺-N/（mg·kg^-1）	硝态氮 NO₃^-N/（mg·kg^-1）	有效磷 AP/（mg·kg^-1）	有效钾 AK/（mg·kg^-1）	总氮 TN/%	总碳 TP/%
稻壳炭RB	9.76±0.34a	4.20±0.32a	1.95±0.11b	35.71±6.12a	854.59±23.11a	1.10±0.86b	0.12±0.05b	1.99±1.02b
烟杆炭TB	10.30±0.24a	0.34±0.11b	3.34±0.18a	7.98±2.54b	743.60±32.14b	8.49±1.22a	0.88±0.13a	62.3±5.21a

表2 三七连作土壤的理化性质和土壤酶活性

Table 2 Physicochemical properties and enzyme activities of soil for continuously cropping P. notoginseng

处理Treatment	对照CK	稻壳炭RB	烟杆炭TB
成活率Survical rate/%	5.93±2.38b	17.17±1.15a	18.17±2.52a
pH	5.98±0.13b	6.38±0.23a	6.34±0.18a
铵态氮NH₄⁺-N/（mg·kg^-1）	3.19±0.11a	2.30±0.55b	2.77±0.12b
硝态氮NO₃^--N/（mg·kg^-1）	7.63±0.44b	7.7±0.19b	29.29±3.34a
有效磷AP/（mg·kg^-1）	7.492±1.10b	15.978±3.10a	16.686±2.66a
有效钾AK/（mg·kg^-1）	138.421±21.05a	137.564±18.04a	124.367±8.32a
微生物量碳MC/（mg·kg^-1）	42.46±6.16a	56.22±7.19a	61.17±23.33a
酸性磷酸酶Phosphatase/[mg·（g·24 h）^-1]	0.190±0.04b	0.150±0.08b	0.550±0.15a
脲酶 Urease/[mg·（g·24 h）^-1]	0.459±0.07b	0.467±0.09b	0.594±0.18a
蔗糖酶Sucrase/[mg·（g·24 h）^-1]	45.910±6.45a	23.475±6.55b	18.217±7.36b

图1 三七成活率与土壤理化性质及酶活性相关性描述分析 NH4+-N：铵态氮；NO3--N：硝态氮；AP：有效磷；AK：有效钾；MC：微生物量碳；Sucrase：蔗糖酶；Phosphatase：磷酸酶；Urease：脲酶。*表示P<0.05，**表示P<0.01。下同

Fig. 1 Descriptive analysis on the correlation between P. notoginseng's survival rates and soil physicochemical properties and enzyme activities NH4+-N: Ammonium nitrogen. NO3--N: Nitrate nitrogen. AP: Available phosphorus. AK: Available potassium. MC: Microbial biomass carbon.）. * indicates P<0.05，and **indicates P<0.01. The same below

图2 属水平不同处理间真菌类群的相对丰度及其与三七成活率的皮尔逊相关系数 R值代表真菌的相对丰度与三七成活率的相关系数

Fig. 2 Abundance of fungal population among different treatments at the genus level in the rhizosphere soil and their Pearson’s correlation coefficients with P. notoginseng’s survival rates R refers to the coefficient between the relative abundance and P. notoginseng’s survival rate

表3 三七连作土壤真菌群落alpha多样性分析

Table 3 Alpha diversity analysis of fungal community in soil for continuous cropping of P. notoginseng

处理Treatment	OTUs	Chao1	Simpson	Shannon
CK	781.4±7.05b	998.24±5.17b	0.8844±0.0194b	4.32±0.93c
稻壳炭RB	776.4±10.95b	950.79±28.98c	0.8993±0.0013b	6.36±0.21b
烟杆炭TB	819.6±10.5a	1044.45±17.69a	0.9950±0.0036a	8.21±0.60a

图3 土壤理化性质与真菌群落相对丰度及多样性指数相关性分析热图

Fig. 3 Heatmap of correlation analysis between soil physi-cochemical properties and fungal community diver-sity index or relative abundance of soil fungal com-munity

图4 基于门（A）与属（B）水平土壤真菌群落结构与土壤理化性质间的冗余分析

Fig. 4 Redundancy analysis between the soil fungal community and soil physicochemical properties based on phylum（A）and genus（B）level

图5 土壤性质及真菌群落指标对三七苗成活率影响的结构模型箭头附近的数字表示标准化路径系数；绿色代表正面贡献，红色代表负面贡献；粗线条代表具有显著相关性。Ascomycota：子囊菌门；Basidiomycota：担子菌门；Basidiomycota：担子菌门；***：P<0.001

Fig. 5 Structural model of the influence of soil properties and fungal community indicators on the survival rate of P. notoginseng seedlings The SEM was conducted with Amos24. The number near the arrow represents the standardized path coefficient; green represents positive contribution, and red represents negative contribution. Bold lines represent significant correlation. ***: P<0.001

参考文献 48

[1]	黄依丹, 成嘉欣, 石颖, 等. 近五年三七化学成分、色谱分析、三七提取物和药理活性的研究进展[J]. 中国中药杂志, 2022, 47(10): 2584-2596.
	Huang YD, Cheng JX, Shi Y, et al. Panax notoginseng: a review on chemical components, chromatographic analysis, P. notoginseng extracts, and pharmacology in recent five years[J]. China J Chin Mater Med, 2022, 47(10): 2584-2596.
[2]	Fan ZY, Miao CP, Qiao XG, et al. Diversity, distribution, and antagonistic activities of rhizobacteria of Panax notoginseng[J]. J Ginseng Res, 2016, 40(2): 97-104. doi: 10.1016/j.jgr.2015.05.003 URL
[3]	邢娜, 彭东辉, 张志宏, 等. 炮制对三七化学成分及药理作用影响的研究进展[J]. 中国实验方剂学杂志, 2020, 26(16): 210-217.
	Xing N, Peng DH, Zhang ZH, et al. Progress in research on effect of processing on chemical constituents and pharmacological effect of notoginseng Radix et rhizoma[J]. Chin J Exp Tradit Med Formulae, 2020, 26(16): 210-217.
[4]	崔秀明, 黄璐琦, 郭兰萍, 等. 中国三七产业现状及发展对策[J]. 中国中药杂志, 2014, 39(4): 553-557.
	Cui XM, Huang LQ, Guo LP, et al. Chinese Sanqi industry status and development countermeasures[J]. China J Chin Mater Med, 2014, 39(4): 553-557.
[5]	孙雪婷, 李磊, 龙光强, 等. 三七连作障碍研究进展[J]. 生态学杂志, 2015, 34(3): 885-893.
	Sun XT, Li L, Long GQ, et al. The progress and prospect on consecutive monoculture problems of Panax notoginseng[J]. Chin J Ecol, 2015, 34(3): 885-893.
[6]	毛忠顺, 龙月娟, 朱书生, 等. 三七根腐病研究进展[J]. 中药材, 2013, 36(12): 2051-2054.
	Mao ZS, Long YJ, Zhu SS, et al. The progress and prospect on root rot of Panax notoginseng[J]. J Chin Med Mater, 2013, 36(12): 2051-2054.
[7]	Itoo ZA, Reshi ZA. The multifunctional role of ectomycorrhizal associations in forest ecosystem processes[J]. Bot Rev, 2013, 79(3): 371-400. doi: 10.1007/s12229-013-9126-7 URL
[8]	Shi LL, Dossa GGO, Paudel E, et al. Changes in fungal communities across a forest disturbance gradient[J]. Appl Environ Microbiol, 2019, 85(12): e00080-e00019.
[9]	韩世忠, 高人, 马红亮, 等. 建瓯万木林自然保护区两种森林类型土壤真菌多样性[J]. 生态学杂志, 2015, 34(9): 2613-2620.
	Han SZ, Gao R, Ma HL, et al. Soil fungal diversities of two types of forests in Jian'ou Wanmulin Nature Reserve[J]. Chin J Ecol, 2015, 34(9): 2613-2620.
[10]	梁雪, 李永春, 陈相君, 等. 新疆木垒胡杨林区三种林分土壤真菌群落特征[J]. 生态学杂志, 2017, 36(3): 623-630.
	Liang X, Li YC, Chen XJ, et al. Soil fungal community characteristics in three types of forest stand in Huyang forest region of Mulei, Xinjiang[J]. Chin J Ecol, 2017, 36(3): 623-630.
[11]	Sugiyama A, Ueda Y, Zushi T, et al. Changes in the bacterial community of soybean rhizospheres during growth in the field[J]. PLoS One, 2014, 9(6): e100709.
[12]	Luo XS, Fu XQ, Yang Y, et al. Microbial communities play important roles in modulating paddy soil fertility[J]. Sci Rep, 2016, 6: 20326. doi: 10.1038/srep20326 pmid: 26841839
[13]	Wu ZX, Hao ZP, Sun YQ, et al. Comparison on the structure and function of the rhizosphere microbial community between healthy and root-rot Panax notoginseng[J]. Appl Soil Ecol, 2016, 107: 99-107. doi: 10.1016/j.apsoil.2016.05.017 URL
[14]	Ma L, Cao YH, Cheng MH, et al. Phylogenetic diversity of bacterial endophytes of Panax notoginseng with antagonistic characteristics towards pathogens of root-rot disease complex[J]. Antonie Van Leeuwenhoek, 2013, 103(2): 299-312. doi: 10.1007/s10482-012-9810-3 URL
[15]	Jiang JL, Yu M, Hou RP, et al. Changes in the soil microbial community are associated with the occurrence of Panax quinquefolius L. root rot diseases[J]. Plant Soil, 2019, 438(1/2): 143-156. doi: 10.1007/s11104-018-03928-4 URL
[16]	Li J, Wang RF, Zhou Y, et al. Dammarane-type triterpene oligoglycosides from the leaves and stems of Panax notoginseng and their antiinflammatory activities[J]. J Ginseng Res, 2019, 43(3): 377-384. doi: 10.1016/j.jgr.2017.11.008 URL
[17]	Li CJ, Li DF, Zhou GS, et al. Effects of different types of biochar on soil microorganism and rhizome diseases occurrence of flue-cured tobacco[J]. Acta Agron Sin, 2019, 45(2): 289. doi: 10.3724/SP.J.1006.2019.01105 URL
[18]	Dangi S, Gao SD, Duan YH, et al. Soil microbial community structure affected by biochar and fertilizer sources[J]. Appl Soil Ecol, 2020, 150: 103452. doi: 10.1016/j.apsoil.2019.103452 URL
[19]	Lehmann J, Rillig MC, Thies J, et al. Biochar effects on soil biota-A review[J]. Soil Biol Biochem, 2011, 43(9): 1812-1836. doi: 10.1016/j.soilbio.2011.04.022 URL
[20]	Beesley L, Moreno-Jiménez E, Gomez-Eyles JL, et al. A review of biochars'potential role in the remediation, revegetation and restoration of contaminated soils[J]. Environ Pollut, 2011, 159(12): 3269-3282. doi: 10.1016/j.envpol.2011.07.023 pmid: 21855187
[21]	Lian F, Liu XW, Gao ML, et al. Effects of Fe-Mn-Ce oxide-modified biochar on As accumulation, morphology, and quality of rice(Oryza sativa L.)[J]. Environ Sci Pollut Res Int, 2020, 27(15): 18196-18207. doi: 10.1007/s11356-020-08355-6 URL
[22]	Palansooriya KN, Wong JTF, Hashimoto Y, et al. Response of microbial communities to biochar-amended soils: a critical review[J]. Biochar, 2019, 1(1): 3-22. doi: 10.1007/s42773-019-00009-2 URL
[23]	牟苗. 生物质土壤改良材料制备及其在三七栽培上的应用[D]. 昆明: 云南师范大学, 2018.
	Mou M. Production and application of improved biomass material in Panax notoginseng cultivation[D]. Kunming: Yunnan Normal University, 2018.
[24]	王昆艳, 王豪吉, 李双丽, 等. 施加生物炭对三七连作土壤铅有效态含量的影响[J]. 云南师范大学学报: 自然科学版, 2019, 39(5): 53-57.
	Wang KY, Wang HJ, Li SL, et al. Effect of biochar application on the content of available lead in Sanqi continuous cropping land[J]. J Yunnan Norm Univ Nat Sci Ed, 2019, 39(5): 53-57.
[25]	Smalla K, Wieland G, Buchner A, et al. Bulk and rhizosphere soil bacterial communities studied by denaturing gradient gel electrophoresis: plant-dependent enrichment and seasonal shifts revealed[J]. Appl Environ Microbiol, 2001, 67(10): 4742-4751. doi: 10.1128/AEM.67.10.4742-4751.2001 URL
[26]	鲍士旦. 土壤农化分析[M]. 3版. 北京: 中国农业出版社, 2000.
	Bao SD. Soil and Agricultural Chemistry Analysis[M]. 3rd ed. Beijing: Chinese Agriculture Press, 2000.
[27]	Vance ED, Brookes PC, Jenkinson DS. An extraction method for measuring soil microbial biomass C[J]. Soil Biol Biochem, 1987, 19(6): 703-707. doi: 10.1016/0038-0717(87)90052-6 URL
[28]	石春芳, 王志勇, 冷小云, 等. 土壤磷酸酶活性测定方法的改进[J]. 实验技术与管理, 2016, 33(7): 48-49, 54.
	Shi CF, Wang ZY, Leng XY, et al. Improvement of soil phosphatase activity measurement method[J]. Exp Technol Manag, 2016, 33(7): 48-49, 54.
[29]	姚槐应, 黄昌勇. 土壤微生物生态学及其实验技术[M]. 北京: 科学出版社, 2006.
	Yao HY, Huang CY. Soil microbial ecology and its experimental techniques[M]. Beijing: Science Press, 2006.
[30]	赵林艳, 官会林, 向萍, 等. 白及根腐病植株根际土壤微生物群落组成特征分析[J]. 生物技术通报, 2022, 38(2): 67-74.
	Zhao LY, Guan HL, Xiang P, et al. Composition features of microbial community in the rhizospheric soil of Bletilla striata with root rot[J]. Biotechnol Bull, 2022, 38(2): 67-74.
[31]	Magoč T, Salzberg SL. FLASH: fast length adjustment of short reads to improve genome assemblies[J]. Bioinformatics, 2011, 27(21): 2957-2963. doi: 10.1093/bioinformatics/btr507 pmid: 21903629
[32]	Bolger AM, Lohse M, Usadel B. Trimmomatic: a flexible trimmer for Illumina sequence data[J]. Bioinformatics, 2014, 30(15): 2114-2120. doi: 10.1093/bioinformatics/btu170 pmid: 24695404
[33]	Edgar RC, Haas BJ, Clemente JC, et al. UCHIME improves sensitivity and speed of chimera detection[J]. Bioinformatics, 2011, 27(16): 2194-2200. doi: 10.1093/bioinformatics/btr381 pmid: 21700674
[34]	Edgar RC. UPARSE: highly accurate OTU sequences from microbial amplicon reads[J]. Nat Methods, 2013, 10(10): 996-998. doi: 10.1038/nmeth.2604 pmid: 23955772
[35]	Schloss PD, Westcott SL, Ryabin T, et al. Introducing mothur: open-source, platform-independent, community-supported software for describing and comparing microbial communities[J]. Appl Environ Microbiol, 2009, 75(23): 7537-7541. doi: 10.1128/AEM.01541-09 URL
[36]	史思伟, 娄翼来, 杜章留, 等. 生物炭的10年土壤培肥效应[J]. 中国土壤与肥料, 2018(6): 16-22.
	Shi SW, Lou YL, Du ZL, et al. A 10-year field experiment on biochar amendment: effects on soil fertility[J]. Soil Fertil Sci China, 2018(6): 16-22.
[37]	赵牧秋, 金凡莉, 孙照炜, 等. 制炭条件对生物炭碱性基团含量及酸性土壤改良效果的影响[J]. 水土保持学报, 2014, 28(4): 299-303, 309.
	Zhao MQ, Jin FL, Sun ZW, et al. Effects of pyrolysis condition on basic group of biochar and amelioration of acid soil[J]. J Soil Water Conserv, 2014, 28(4): 299-303, 309.
[38]	Xu WM, Wu FY, Wang HJ, et al. Key soil parameters affecting the survival of Panax notoginseng under continuous cropping[J]. Sci Rep, 2021, 11(1): 5656. doi: 10.1038/s41598-021-85171-z URL
[39]	张家春, 孙超, 李朝桢, 等. 不同种植年限白及土壤有机质、酶活性与白及有效成分研究[J]. 中药材, 2020, 43(1): 1-4.
	Zhang JC, Sun C, Li CZ, et al. Study on soil organic matter, enzyme activity and the effective components of Bletilla striata under different planting years[J]. J Chin Med Mater, 2020, 43(1): 1-4.
[40]	Dong LL, Xu J, Feng GQ, et al. Soil bacterial and fungal community dynamics in relation to Panax notoginseng death rate in a continuous cropping system[J]. Sci Rep, 2016, 6: 31802. doi: 10.1038/srep31802 URL
[41]	文东新, 杨宁, 杨满元. 衡阳紫色土丘陵坡地植被恢复对土壤微生物功能多样性的影响[J]. 应用生态学报, 2016, 27(8): 2645-2654. doi: 10.13287/j.1001-9332.201608.009
	Wen DX, Yang N, Yang MY. Effects of re-vegetation on soil microbial functional diversity in purple soils at different revegetation stages on sloping-land in Hengyang, Hunan Province, China[J]. Chin J Appl Ecol, 2016, 27(8): 2645-2654.
[42]	李永赟, 刘羽佳, 叶田会, 等. 攀枝花不同海拔植烟土壤固氮细菌群落多样性与结构特征[J]. 应用与环境生物学报, 2022, 28(6): 1452-1459.
	Li YY, Liu YJ, Ye TH, et al. Diversity and structural characteristics of nitrogen-fixing bacterial community in tobacco-growing soils at different elevations in Panzhihua[J]. Chin J Appl Environ Biol, 2022, 28(6): 1452-1459.
[43]	金鑫, 李孝敬, 黄艺伟, 等. 不同年份三七根际土壤中真菌的分离鉴定及其多样性的比较[J]. 中药材, 2020, 43(2): 314-317.
	Jin X, Li XJ, Huang YW, et al. solation and identification of fungi in rhizosphere soil of Panax notoginseng in different years and comparison of their diversity[J]. J Chin Med Mater, 2020, 43(2): 314-317.
[44]	Gardes M, Bruns TD. ITS primers with enhanced specificity for basidiomycetes—application to the identification of mycorrhizae and rusts[J]. Mol Ecol, 1993, 2(2): 113-118. doi: 10.1111/j.1365-294x.1993.tb00005.x pmid: 8180733
[45]	代红翠, 张慧, 薛艳芳, 等. 不同耕作和秸秆还田下褐土真菌群落变化特征[J]. 中国农业科学, 2019, 52(13): 2280-2294. doi: 10.3864/j.issn.0578-1752.2019.13.008
	Dai HC, Zhang H, Xue YF, et al. Response of fungal community and function to different tillage and straw returning methods[J]. Sci Agric Sin, 2019, 52(13): 2280-2294. doi: 10.3864/j.issn.0578-1752.2019.13.008
[46]	宁琪, 陈林, 李芳, 等. 被孢霉对土壤养分有效性和秸秆降解的影响[J]. 土壤学报, 2022, 59(1): 206-217.
	Ning Q, Chen L, Li F, et al. Effects of Mortierella on nutrient availability and straw decomposition in soil[J]. Acta Pedol Sin, 2022, 59(1): 206-217.
[47]	Tan Y, Cui YS, Li HY, et al. Rhizospheric soil and root endogenous fungal diversity and composition in response to continuous Panax notoginseng cropping practices[J]. Microbiol Res, 2017, 194: 10-19. doi: 10.1016/j.micres.2016.09.009 URL
[48]	黄宇, 汪汉成, 陈乾丽, 等. 感染白粉病烟株叶片真菌的群落结构与多样性[J]. 贵州农业科学, 2020, 48(5): 54-58.
	Huang Y, Wang HC, Chen QL, et al. Community structure and diversity of fungi in leaves of tobacco plant infected with powdery mildew[J]. Guizhou Agric Sci, 2020, 48(5): 54-58.