韭菜根际真菌和内生真菌种群多样性分析

doi:10.7668/hbnxb.2018.S1.039

摘要/Abstract

摘要： 在前期研究发现，韭菜处理可以减轻香蕉枯萎病和根结线虫病害的发生，推测韭菜中含有能抑制病原菌及致死根结线虫的生防菌。对韭菜根际真菌和内生（叶片和根系）真菌的种群结构及多样性进行比较分析，以期为分离鉴定韭菜中抑菌和杀根结线虫的生防真菌提供理论依据。通过Illumina平台对韭菜根际真菌和内生（叶片和根系）真菌样品中的18S rRNA序列进行测序，并进行生物信息学分析。结果表明：从韭菜根际土壤、根系、叶片中获得348 484高质量序列和123个操作分类单元（OTU）。根际真菌的Chao、ACE、Shannon指数显著高于内生真菌（叶片和根系），而Simpson指数则显著小于内生真菌。根际真菌群落主要由子囊菌门（Ascomycota）（67.20%）和担子菌门（Basidiomycota）（24.44%）2个菌门组成；而内生真菌主要由子囊菌门组成，在叶片和根系中含量分别为91.39%和96.60%。在检测到的5个菌门中，内生真菌中子囊菌门显著高于根际土壤，其他菌门则显著低于根际土壤。叶片和根系中内生真菌稍有不同，但是差异并不显著。该研究表明，韭菜根际真菌和内生真菌的种群结构存在显著差异，根际真菌的多样性及丰富性显著高于内生真菌。该研究为分离鉴定韭菜中抑制植物病原菌及致死根结线虫的生防真菌提供参考。

关键词: 韭菜, 真菌多样性, Illumina测序, 内生菌, 根际土壤

Abstract: In the preliminary study,the incidence of Fusarium wilt of banana and the disease caused by root-knot nematode were significantly reduced by Chinese leek. It was deduced that some potential biological fungus with antifungal and nemotocidal activity existed in the Chinese leek. In the present study the rhizospheric and endophytic fungus communities and diversities were comparatively analyzed,aiming to provide theoretical basis for isolating and identifying biological fungus with antifungal and nematocidal activity in Chinese leek. 18S rRNA from the samples of Chinese leek rhizosphere soil,leave and root were sequenced on the Illumina Miseq platform,and the datas were comparatively analyzed using bioinformatics. The results showed a total of 348 484 high quality sequences and 123 operational taxonomic units(OTUs) was obtained from the Chinese leek rhizosphere siol, leave and root. The Chao,ACE,Shannon index of rhizospheric fungus were significantly higher and Simpson index was significantly lower than that of endophytic fungus(leave and root). The rhizopheric fungus was mainly composed by Ascomycota(67.20%) and Basidiomycota(24.44%). The endophytic fungus was mainly composed by Ascomycota,91.39% in leave and 96.60% in root. In the identified five fungus phyla,the amount of Ascomycota in endophytic fungus was significantly higher than that in the rhizophytic fungus,the other fungus phyla was significantly lower than that in the rhizophytic fungus. The fungus community in the leave and root was slightly different,but not significant. The present study showed significantly difference in the rhizophytic and endophytic fungus communities. The diversity and richness of rhizophytic fungus were significantly higher than that of endophytic fungus. The findings of the study provide reference to isolate biological fungus with potential antifungal and nematocidal activity from Chinese leek.

Key words: Chinese leek, Fungal diversity, Illumina sequence, Endophyte, Rhizophyere soil

中图分类号:

S432

付静, 刘堰凤, 梅眉, 黄永红. 韭菜根际真菌和内生真菌种群多样性分析[J]. 华北农学报, 2018, 33(S1): 246-252. doi: 10.7668/hbnxb.2018.S1.039.

FU Jing, LIU Yanfeng, MEI Mei, HUANG Yonghong. Comparing Analysis of Diversity of Chinese Leek Rhizospheritic and Endophytic Fungal Communities[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2018, 33(S1): 246-252. doi: 10.7668/hbnxb.2018.S1.039.

http://www.hbnxb.net/CN/Y2018/V33/IS1/246

参考文献

[1] 陆雅海,张福锁. 根际微生物研究进展[J]. 土壤,2006,38(2):113-121.
[2] Murphy B R,Doohan F M,Hodkinson T R. Fungal root endophytes of a wild barley species increase yield in a nutrient-stressed barley cultivar[J]. Symbiosis,2015,65(1):1-7.
[3] 姜道宏. 植物内生真菌及其展望[J]. 中国生物防治学报,2015,31(5):742-749.
[4] 赵龙飞,徐亚军,常佳丽. 轮纹病菌拮抗性大豆根瘤内生菌的筛选、抗性和促生作用[J]. 中国生物防治学报,2016,32(3):396-405.
[5] Khan A L,Waqas M,Hussain J,et al. Endophytes Aspergillus caespitosus LK12 and Phoma sp.LK13 of Moringa peregrina produce gibberellins and improve rice plant growth[J]. Journal of Plant Interactions,2014,9(1):731-737.
[6] Murphy B R,Doohan F M,Hodkinson T R. Persistent fungal root endophytes isolated from a wild barley species suppress seed-borne infections in a barley cultivar[J]. BioControl,2015,60(2):281-292.
[7] 高春梅,李敏,刘润进. AMF和DSE组合菌剂促生防线虫病效应[J]. 菌物学报,2016,35(10):1208-1217.
[8] Huang Y H,Wang R C,Li C H,et al. Control of fusarium wilt in banana with Chinese leek[J]. European Journal of Plant Pathology,2012,134(1):87-95.
[9] Huang Y H, Mao Z C, Xie B Y. Chinese leek(Allium tuberosum Rottler ex Sprengel) reduced disease symptom caused by root-knot nematode[J]. Journal of Integrative Agriculture,2016,15(2):364-372.
[10] Zuo C W,Li C Y,Li B,et al. The toxic mechanism and bioactive components of Chinese leek root exudates acting against Fusarium oxysporum f. sp cubense tropical race 4[J]. European Journal of Plant Pathology,2015,143(3):447-460.
[11] 黄永红.韭菜对根结线虫的防控作用及其活性成分分析研究[D].北京:中国农业科学院, 2015.
[12] 黄永红,吕顺,李春雨,等. 香蕉枯萎病菌4号生理小种对香蕉根际土壤微生物及酶活性的影响[J]. 湖南农业大学学报:自然科学版,2012,38(2):173-176.
[13] Huang Y H. Comparison of rhizosphere and endophytic microbial communities of Chinese leek through high-throughput 16S rRNA gene Illumina sequencing[J]. Journal of Integrative Agriculture,2018,17(2):359-367.
[14] Tasumi E,Kanazawa S,Fukuda S. Degradation of a herbicide,beflubutamid,in upland soils and isolation of its degrading microbes[J]. Soil Science & Plant Nutrition,2007,53(3):236-245.
[15] Smit E,Leeflang P,Glandorf B,et al. Analysis of fungal diversity in the wheat rhizosphere by sequencing of cloned PCR-amplified genes encoding 18S rRNA and temperature gradient gel electrophoresis[J]. Applied & Environmental Microbiology,1999,65(6):2614-2621.
[16] Schloss P D,Westcott S L,Ryabin T,et al. Introducing mothur:open-source,platform-independent,community-supported software for describing and comparing microbial communities[J]. Applied and Environmental Microbiology,2009,75(23):7537-7541.
[17] Edgar R C. UPARSE:highly accurate OTU sequences from microbial amplicon reads[J]. Nature Methods,2013,10(10):996-998.
[18] Wang Q,Garrity G M,Tiedje J M,et al. Na?ve bayesian classifier for rapid assignment of rRNA sequences into the new bacterial taxonomy[J]. Applied & Environmental Microbiology,2007,73(16):5261-5267.
[19] Pruesse E,Quast C,Knittel K,et al. SILVA:a comprehensive online resource for quality checked and aligned ribosomal RNA sequence data compatible with ARB[J]. Nucleic Acids Research,2007,35(21):7188-7196.
[20] Caporaso J G,Bittinger K,Bushman F D,et al. PyNAST:a flexible tool for aligning sequences to a template alignment[J]. Bioinformatics,2010,26(2):266-267.
[21] Price M N,Dehal P S,Arkin A P. FastTree:computing large minimum evolution trees with profiles instead of a distance matrix[J]. Molecular Biology and Evolution,2009,26(7):1641-1650.
[22] Lozupone C,Knight R. UniFrac:a new phylogenetic method for comparing microbial communities[J]. Applied and Environmental Microbiology,2005,71(12):8228-8235.
[23] White J R,Nagarajan N,Pop M. Statistical methods for detecting differentially abundant features in clinical metagenomic samples[J]. PLoS Computational Biology,2009,5(4):e1000352.
[24] 宋伦,刘卫东,吴景,等. 有害甲藻Stoeckeria algicida 在辽东湾的时空分布[J]. 生态学报,2017,37(4):1339-1345.
[25] 王雪庆,于淑惠,孙涛,等. 白蜡虫蜡花微生物多样性分析[J]. 昆虫学报,2016,59(10):1086-1092.
[26] Jia H R,Geng L L,Li Y H,et al. The effects of Bt Cry1le toxin on bacterial diversity in the midgut of Apis mellifera ligustica(Hymenoptera:Apidae)[J]. Scientific Reports,2016,6:24664).
[27] Guo J J,Fu X L,Liao H D,et al. Potential use of bacterial community succession for estimating post-mortem interval as revealed by high-throughput sequencing[J]. Scientific Reports,2016,6:24197.
[28] Jiao S,Liu Z S,Lin Y B,et al. Bacterial communities in oil contaminated soils:Biogeography and co-occurrence patterns[J]. Soil Biology & Biochemistry,2016,98:64-73.
[29] Wang J,Xue C,Song Y,et al. Wheat and rice growth stages and fertilization regimes alter Soil bacterial community structure,but not diversity[J]. Frontiers in Microbiology,2016,7:1207.
[30] Fang H,Lian J J,Wang H F,et al. Exploring bacterial community structure and function associated with atrazine biodegradation in repeatedly treated soils[J]. Journal of Hazardous Materials,2015,286:457-465.
[31] 陈丹梅,陈晓明,梁永江,等. 种植模式对土壤酶活性和真菌群落的影响[J]. 草业学报,2015,24(2):77-84.
[32] Shen Z Z,Ruan Y Z,Chao X E,et al. Rhizosphere microbial community manipulated by 2 years of consecutive biofertilizer application associated with banana Fusarium wilt disease suppression[J]. Biology and Fertility of Soils,2015,51(5):553-562.
[33] Yu L,Nicolaisen M,Larsen J,et al. Molecular characterization of root-associated fungal communities in relation to health status of Pisum sativum using barcoded pyrosequencing[J]. Plant and Soil,2012,357(1-2):395-405.
[34] Bais H P,Weir T L,Perry L G,et al. The role of root exudates in rhizosphere interactions with plants and other organisms[J]. Annual Review of Plant Biology,2006,57:233-266.
[35] Shen Z Z,Wang D S,Ruan Y Z,et al. Deep 16S rRNA pyrosequencing reveals a bacterial community associated with banana fusarium wilt disease suppression induced by Bio-Organic fertilizer application[J]. PLoS One,2014,9(5):e98420.
[36] Koukol O,Kolarik M,Kolarova Z,et al. Diversity of foliar endophytes in wind-fallen Picea abies trees[J]. Fungal Diversity,2012,54(1):69-77.
[37] Compant S,Mitter B,Gualberto Colli-Mull J A,et al. Endophytes of grapevine flowers,berries,and seeds:identification of cultivable bacteria,comparison with other plant parts,and visualization of niches of colonization[J]. Microbial Ecology,2011,62(1):188-197.
[38] Masuko K,Murata M,Yudoh K,et al. Ion torrent PGM as Tool for fungal community analysis:a case study of endophytes in eucalyptus grandis reveals high taxonomic diversity[J]. PLoS One,2013,8(12):e81718.
[39] 赵爽,张宁,刘东阳,等. 氯苯嘧啶醇施用对草坪斑枯病致病菌及根际土壤真菌的影响[J]. 南京农业大学学报,2015,38(4):590-595.
[40] Wu X,Zhang H,Chen J,et al. Comparison of the fecal microbiota of dholes high-throughput Illumina sequencing of the V3-V4 region of the 16S rRNA gene[J]. Applied Microbiology and Biotechnology,2016,100(8):3577-3586.

选择文件类型/文献管理软件名称

选择包含的内容