石灰对灵芝覆土连作障碍的防控作用及其微生物群落分析

doi:10.13560/j.cnki.biotech.bull.1985.2020-0786

摘要/Abstract

摘要：

揭示石灰处理连作覆土对灵芝出芝率、产量、病害率和微生物群落变化的影响,为灵芝连作障碍的防控措施提供理论依据。以连作4年的灵芝覆土（CK）为对照,通过对覆土进行22.5 kg石灰浸泡处理（LI45）、45 kg石灰浸泡处理（LI90）和45 kg石灰匀撒处理（LS90）,进而采用Illumina NovaSeq平台分别对CK、LI45和LI90组进行ITS1和16S rDNA扩增子测序。结果发现,相同剂量下石灰浸泡处理比石灰匀撒处理连作覆土显著改善灵芝连作障碍,并且在石灰浸泡处理组中出芝率、产量和部分菌群丰度变化呈剂量依赖性。与对照组相比,LI90组作用效果最显著,出芝率约提高了68.89%,产量约提高了135.87%,病害率大约降低了54.90%;在真菌属水平上,LI90组子囊菌门曲霉属等相对丰度较对照组明显降低;在细菌属水平上,LI90组覆土中贪铜菌属和嗜酸菌属等相对丰度较对照组显著提高,且贪铜菌属和嗜酸菌属相对丰度呈剂量递增。综上,LI90组石灰浸泡处理能够削减灵芝连作障碍作用,其可能与灵芝连作覆土中曲霉属等病原菌的降低,以及贪铜菌属等多种有益菌的增加有关。

关键词: 灵芝, 连作障碍, 石灰, 扩增子测序, 微生物群落

Abstract:

The effects of lime treatment on the emergence rate of Ganoderma lingzhi,yield,disease rate and microbial community of G. lingzhi were revealed,which may provide theoretical basis for the prevention and control measures of G. lingzhi continuous cropping obstacles. Taking 4-years continuous cropping soil of G. lingzhi as control（CK）,the soil respectively was treated with 22.5 kg lime soaked（LI45）,45 kg lime soaked（LI90）and 45 kg lime scattered（LS90）,then Illumina NovaSeq platform was used to sequence the ITS1 and 16S rDNA amplicons of group CK,LI45 and LI90 respectively. The results showed that the lime soaking treatment at the same dose significantly improved the continuous cropping obstacles of G. lingzhi than the lime scattering treatment,and the change rates of G. lingzhi emergence,yield and abundance of partial microbial communities in the lime soaking treatment were dose-dependent. The effect in the LI90 was the most significant while compared with the CK,the rate of G. lingzhi emergence approximately increased by 68.89%,the yield approximately increased by 135.87%,and the disease rate approximately decreased by 54.90%. Comparing with the CK,the relative abundance of Ascomycota Aspergillus in the LI90 significantly reduced at the fungal genus level. At the bacterial genus level,the relative abundance of Proteobacteria Cupriavidus and Acidovorax were significantly higher in the soil of LI90 than that of the CK,and the relative abundances of Cupriavidus and Acidovorax increased in a doses-dependent manner. In conclusion,the continuous cropping barrier effect of G. lingzhi can be reduced by LI90 treatment,which may be related to the reduction of pathogens such as Aspergillus in continuous cropping soil,and the increase of various beneficial bacteria such as Cupriavidus.

Key words: Ganoderma lingzhi, continuous cropping obstacle, lime, amplicon sequencing, microbial community

袁源, 黄海辰, 李琳, 刘国辉, 傅俊生, 吴小平. 石灰对灵芝覆土连作障碍的防控作用及其微生物群落分析[J]. 生物技术通报, 2021, 37(4): 70-84.

YUAN Yuan, HUANG Hai-chen, LI Lin, LIU Guo-hui, FU Jun-sheng, WU Xiao-ping. Effect of Lime on Preventing and Controlling Continuous Cropping Obstacle of Ganoderma lingzhi and Analysis of Its Microbial Community[J]. Biotechnology Bulletin, 2021, 37(4): 70-84.

图/表 12

表1 不同石灰处理方式

Table 1 Different lime treatment methods

分组Group	处理日期Date of treatment	大棚编号No. of greenhouse	处理方法Methods
CK	2019年2月27日	6号大棚	面积为150 m²大棚翻耕覆土,不进行任何处理
LI45		1号大棚	面积为150 m²大棚匀撒22.5 kg石灰,灌水浸泡7 d
LI90		2号大棚	面积为150 m²大棚匀撒45 kg石灰,灌水浸泡7 d
LS90		3号大棚	面积为150 m²大棚匀撒45 kg石灰,翻耕匀拌

图1 灵芝初期生长情况分析 A.出芝率;B.出芝期灵芝生长状态。与对照组相比,*P<0.05差异显著

Fig.1 Analysis of initial growth of G. lingzhi A: Rate of G. lingzhi emergence, B: Growth status of G. lingzhi at emergence. Significant difference: *P<0.05 compared to control group

图2 灵芝后期生长情况分析 A.灵芝产量;B.成熟期灵芝生长状态。与对照组相比,**P<0.01差异极显著

Fig.2 Analysis of later growth of G. lingzhi A: Yield of G. lingzhi, B: Growth status of G. lingzhi at maturity. Significant difference extremely: **P<0.01 compared to control group

图3 灵芝病害率统计与分析与对照组相比,*P<0.05差异显著

Fig.3 Statistics and analysis of G. lingzhi disease rate Significant difference: *P<0.05 compared to control group

图4 真菌feature分布维恩图与α和β多样性分析 A:维恩分布图;B:主成分分析;C:观察到的feature数目;D:Simpson指数。与对照组相比,*P<0.05差异显著

Fig.4 Venn diagram of fungal feature distribution and analysis of α and β diversity of fungi A: Venn distribution diagram. B: Principal component analysis. C: Number of features observed. D: Simpson index. Significant difference: *P<0.05 compared to control group

图5 属水平真菌群落的相对丰富度 A.真菌物种分布图;B.物种变化热图。与对照组相比,*P<0.05差异显著,**P<0.01差异极显著

Fig.5 Relative abundance of fungal communities at genus level A: Distribution map of fungi species. B: Heat map of species change. Significant difference: *P<0.05 compared to control group, Significant difference extremely: **P<0.01 compared to control group

图6 覆土中真菌LEfSe物种差异分析 A.多级物种层级树：不同圆圈层从内至外辐射分别代表界门纲目科属种7个分类水平,各个节点代表该水平下的一个物种分类,该物种丰度越高节点越大;B.LDA判别结果

Fig.6 Analysis of the difference of the fungal LEfSe species in the covering soil A: Multi-species hierarchical tree: The radiation from the inside to the outside of the different circle layer refers to seven classification levels of domain, phylum, class, order, family, genus, species respectively, and each node represents a species classification at this level. The higher the abundance of the species, the larger the node. B: LDA discriminant results

图7 覆土中真菌相关性分析 A.网络互作分析图：节点代表覆土中不同的优势属,其大小代表菌群的丰富度,节点之间的连接表明两个优势属之间存在显著的相关性,线条粗细表示相关性的强弱,实线表明呈正相关,虚线表明负相关;B.相关性热图分析

Fig.7 Correlation analysis of fungi in the covering soil A: Network interaction analysis diagram: The nodes represent different dominant genera in the covering soil, and their sizes represent the richness of fungi. The connection between the nodes indicates that there is a significant correlation between the two dominant genera. The thickness of the lines indicates the strength of correlation, the solid lines indicate positive correlation, and the dashed lines indicate negative correlation, B: Correlation analysis of heat map

图8 细菌feature分布维恩图与α和β多样性分析 A:维恩分布图;B:主成分分析;C:观察到的feature数目;D：Simpson指数

Fig.8 Venn diagram of bacterial feature distribution and analysis of α and β diversity of bacteria A: Venn distribution diagram. B: Principal component analysis. C: Number of observed features. D: Simpson index

图9 属水平细菌群落的相对丰富度 A.细菌物种分布图;B.物种变化热图。与对照组相比,**P<0.05差异显著,**P<0.01差异极显著

Fig.9 Relative abundance of bacterial communities at genus level A: Distribution map of bacteria species. B: Heat map of species change. Significant difference: **P<0.05 compared to control group. Significant difference extremely: ***P<0.01 compared to control group

图10 覆土中细菌LEfSe物种差异分析 A：多级物种层级树;B：LDA判别结果

Fig.10 Analysis of the difference of the bacterial LEfSe species in the covering soil A: Multi-species hierarchical tree. B: LDA discriminant results

图11 覆土中细菌相关性分析 A：网络互作分析图;B：相关性热图分析

Fig.11 Correlation analysis of bacteria in the covering soil A: Network interaction analysis diagram. B: Correlation analysis of heat map

参考文献 26

[1]	戴玉成, 杨祝良. 中国药用真菌名录及部分名称的修订[J]. 菌物学报, 2008(6):801-824.
	Dai YC, Yang ZL. A revised checklist of medicinal fungi in China[J]. Mycosystema, 2008(6):801-824.
[2]	宋向文, 张丽, 陈乃富, 等. 大别山区灵芝生产现状与存在问题研究[J]. 现代中药研究与实践, 2015,29(5):4-6, 17.
	Song XW, Zhang L, Chen NF, et al. Review on production status and existing problems of Ganoderma lucidum in dabie mountains[J]. Res Pract Chin Med, 2015,29(5):4-6, 17.
[3]	王明蛟, 高航, 刚婉娇, 等. 灵芝化学成分分析方法的研究[J]. 长春中医药大学学报, 2014,30(5):817-819.
	Wang MJ, Gao H, Gang WJ, et al. Research on the analysis methods for chemical constituents in Ganoderma lucidum[J]. J Changchun Univ Chin Med, 2014,30(5):817-819.
[4]	金鑫, 刘宗敏, 黄羽佳, 等. 我国灵芝栽培现状及发展趋势[J]. 食药用菌, 2016,24(1):33-37.
	Jin X, Liu ZM, Huang YJ, et al. The current situation and development trend of Ganoderma lucidum cultivation in my country[J]. Edib Med Mush, 2016,24(1):33-37.
[5]	吴晓明, 徐文仙, 张树生. 液氨熏蒸土壤防控灵芝连作障碍的效果[J]. 食药用菌, 2018,26(5):322-324.
	Wu XM, Xu WX, Zhang SS. Efficacy evaluation of applying liquid ammonia fumigants to Ganoderma lucidum pathogens-polluted soil[J]. Edib Med Mush, 2018,26(5):322-324.
[6]	马红梅, 李小兵, 符浩, 等. 灵芝连作障碍的土壤微生物种群特性及其生物防治初探[J]. 河南农业科学, 2014,43(3):53-58.
	Ma HM, Li XB, Fu H, et al. Preliminary study on microflora of Ganoderma lucidum continuous cropping obstacle soil and its biological control[J]. Journal of Henan Agricultural Sciences, 2014,43(3):53-58.
[7]	马红梅, 陈永敢, 徐小雄, 等. 灵芝连作障碍的土壤微生态研究[J]. 山东农业大学学报:自然科学版, 2013,44(4):539-542.
	Ma HM, Chen YG, Xu XX, et al. Study on soil microecology of Ganoderma lucidum continuous cropping obstacle[J]. Journal of Shandong Agricultural University:Natural Science Edition, 2013,44(4):539-542.
[8]	袁源, 黄海辰, 叶丽云, 等. 灵芝连作土壤真菌群落分析[J]. 菌物学报, 2019,38(12):2112-2121.
	Yuan Y, Huang HC, Ye LY, et al. Analysis of fungal community in continuous cropping soil of Ganoderma lingzhi[J]. Mycosystema, 2019,38(12):2112-2121.
[9]	Masud MM, Baquy MA, Akhter S, et al. Liming effects of poultry litter derived biochar on soil acidity amelioration and maize growth[J]. Ecotoxicol Environ Safe, 2020,202:110865. doi: 10.1016/j.ecoenv.2020.110865 URL
[10]	曹胜, 欧阳梦云, 周卫军, 等. 石灰对土壤重金属污染修复的研究进展[J]. 中国农学通报, 2018,34(26):109-112.
	Cao S, Ouyang MY, Zhou WJ, et al. Remediation of heavy metal contaminated soils by lime:A review[J]. Chinese Agricultural Science Bulletin, 2018,34(26):109-112.
[11]	闫志浩, 胡志华, 王士超, 等. 石灰用量对水稻油菜轮作区土壤酸度、土壤养分及作物生长的影响[J]. 中国农业科学, 2019,52(23):4285-4295.
	Yan ZH, Hu ZH, Wang SC, et al. Effects of lime content on soil acidity, soil nutrients and crop growth in rice-rape rotation system[J]. Scientia Agricultura Sinica, 2019,52(23):4285-4295.
[12]	唐明, 向金友, 袁茜, 等. 酸性土壤施石灰对土壤理化性质、微生物数量及烟叶产质量的影响[J]. 安徽农业科学, 2015,43(12):91-93.
	Tang M, Xiang JY, Yuan Q, et al. Effects of applying lime on physical and chemical properties, microbial quantities of acid soil and yield and quality of flue-cured tobacco[J]. Journal of Anhui Agricultural Sciences, 2015,43(12):91-93.
[13]	Shen G, Zhang S, Liu X, et al. Soil acidification amendments change the rhizosphere bacterial community of tobacco in a bacterial wilt affected field[J]. Appl Microbiol Biotechnol, 2018,102(22):9781-9791. doi: 10.1007/s00253-018-9347-0 URL
[14]	王一鸣, 赖朝圆, 阮云泽, 等. 石灰联合碳铵熏蒸对连作香蕉生长和根际微生物群落结构的影响[J]. 中国南方果树, 2018,47(3):5-13.
	Wang YM, Lai CY, Ruan YZ, et al. Effects of lime-ammonium bicarbonate fumigation on banana growth and structure of rhizosphere microbial community in a continuous cropping system[J]. South China Fruits, 2018,47(3):5-13.
[15]	米其利, 李雪梅, 管莹, 等. 高通量测序在食品微生物生态学研究中的应用[J]. 食品科学, 2016,37(23):302-308.
	Mi QL, Li XM, Guan Y, et al. Application of high-troughput sequencing in food microbial ecology:a review[J]. Food Science, 2016,37(23):302-308.
[16]	翟婉璐, 钟哲科, 高贵宾, 等. 覆盖经营对雷竹林土壤细菌群落结构演变及多样性的影响[J]. 林业科学, 2017,53(9):133-142.
	Zhai WL, Zhong ZK, Gao GB, et al. Influence of mulching management on soil bacterial structure and diversity in phyllostachys praecox stands[J]. Scientia Silvae Sinicae, 2017,53(9):133-142.
[17]	Schmidt PA, Bálint M, Greshake B, et al. Illumina metabarcoding of a soil fungal community[J]. Soil Biol Biochem, 2013,65:128-132. doi: 10.1016/j.soilbio.2013.05.014 URL
[18]	赵力. 基于高通量测序技术的枯草芽孢杆菌在体动态变化研究[D]. 济南:山东大学, 2017.
	Zhao L. Study on the dynamic changes of Bacillus subtilis in vivo based on high-throughput sequencing technology[D]. Ji’nan:Shandong University, 2017.
[19]	祝宁, 康勇, 齐长红, 等. 大棚草莓避雨基质育苗与灵芝高效轮作栽培模式[J]. 蔬菜, 2019(10):54-56.
	Zhu N, Kang Y, Qi CH, et al. High efficient rotation cultivation mode of sheltered-strawberry seedling and Ganoderma lucidum in greenhouse[J]. Vegetables, 2019(10):54-56.
[20]	马红梅, 陈大雄, 陈永敢. 灵芝连作障碍土壤中的优势微生物种群对灵芝菌丝体的化感作用[J]. 热带作物学报, 2016,37(2):372-375.
	Ma HM, Chen DX, Chen YG. Alelopathic effect of dominant microflora on its mycelium of continuous cropping obstacles Ganoderma lucidum in field cultivation[J]. Chinese Journal of Tropical Crops, 2016,37(2):372-375.
[21]	Baig MN, Shahid AA, Ali M. In vitro assessment of extracts of the Lingzhi or Reishi medicinal mushroom, Ganoderma lucidum(higher Basidiomycetes)against different plant pathogenic fungi[J]. Int J Med Mushrooms, 2015,17(4):407-411. doi: 10.1615/IntJMedMushrooms.v17.i4 URL
[22]	文冬华, 刘善红, 盛立柱, 等. 灵芝覆盖新土的栽培效果试验[J]. 食药用菌, 2017,25(2):125-127.
	Wen DH, Liu SH, Sheng LZ, et al. Cultivation effect test of Ganoderma lucidum covered with new soil[J]. Edible and Medicinal Mushrooms, 2017,25(2):125-127.
[23]	庞师婵, 郭霜, 任奎瑜, 等. 番茄/茄子嫁接对其根际土壤生物学性状及细菌群落结构的影响[J]. 园艺学报, 2020,47(2):253-263.
	Peng SC, Guo S, Ren KY, et al. Impact of grafting on soil microbial properties and bacterial community structure in tomato rhizosphere[J]. Acta Hort Sinica, 2020,47(2):253-263.
[24]	Tiwari J, Naoghare P, Sivanesan S, et al. Biodegradation and detoxification of chloronitroaromatic pollutant by Cupriavidus[J]. Bioresour Technol, 2017,223:184-191. doi: 10.1016/j.biortech.2016.10.043 URL
[25]	Watanabe W, Hori Y, Nishimura S, et al. Bacterial degradation and reduction in the estrogen activity of 4-nonylphenol[J]. Biocontrol Sci, 2012,17(3):143-147. pmid: 23007106
[26]	Cui G, Chien MF, Suto K, et al. Analysis of stable 1, 2-dichlorobenzene-degrading enrichments and two newly isolated degrading strains, Acidovorax sp. sk40 and Ralstonia sp. sk41[J]. Appl Microbiol Biotechnol, 2017,101(17):6821-6828. doi: 10.1007/s00253-017-8406-2 URL