高粱根际土壤细菌群落对盐胁迫的响应

doi:10.13560/j.cnki.biotech.bull.1985.2023-0685

摘要/Abstract

摘要：

【目的】探究盐胁迫下高粱根系微生物群落结构变化，以及高粱根系微生物群落网络特征。【方法】以2种耐盐性不同的高粱品种河农16（盐敏感）和高粱蔗（耐盐）为试验材料，通过盆栽种植，不同盐胁迫处理，利用16S扩增子测序技术对其根系微生物组进行高通量测序。【结果】随着盐胁迫的加剧，高粱根系的总酚和总黄酮含量逐渐增加，适宜的盐胁迫通过诱导酚酸类化合物的合成来提高高粱的耐盐性。高粱根际土壤优势菌门为变形菌门（Proteobacteria）、放线菌门（Actinobacteria）、酸杆菌门（Acidobacteria）、拟杆菌门（Bacteroidetes）和绿弯菌门（Chloroflexi）。盐胁迫条件下Proteobacterisa、Bacteroidetes和Pseudomonas的相对丰度随盐胁迫的加剧显著升高。盐胁迫下高粱根际细菌组成受生育时期的影响较小，高粱根际细菌的群落结构均随盐胁迫程度的加剧而变迁。加权基因共表达相关网络分析（weighted gene co-expression network analysis, WGCNA）鉴定了12个基因共表达模块，其中pink模块与盐胁迫显著正相关，greenyellow模块与生育时期和盐处理后的根系总酚、总黄酮含量显著正相关。低盐土壤细菌共现网络比高盐土壤更为复杂，表现为节点和连接更多。鉴定出13个网络关键OTUs，OTU8480、OTU6866、OTU3247和OTU3499是S0网络中的关键OTUs，S3网络中为OTU6895、OTU4206、OTU6470、OTU1810和OTU4916，S7网络以OTU4217、OTU8426、OTU4847和OTU6066为关键类群。【结论】盐胁迫下高粱根系微生物的多样性发生改变，共现网络更为复杂，OTUs间联系更为紧密。

关键词: 高粱, 根际, 微生物群落结构, 盐胁迫

Abstract:

【Objective】This study aims to investigate the changes in the bacterial community structure of sorghum roots under salt stress, as well as the characteristics of the rhizosphere bacterial co-occurrence network structure.【Method】Two sorghum varieties, HN16（salt-sensitive）and GLZ（salt-tolerant）, with varying levels of salt tolerance were cultivated in pots and subjected to salt stress. The root microbiome of these plants was then analyzed using 16S amplicon sequencing technology.【Result】With the aggravation of salt stress, the contents of total phenols and flavonoids in the sorghum roots gradually increased, and the appropriate salt stress induced the synthesis of phenolic acids to improve the salt tolerance of sorghum. The results of the microbial community structure analysis showed that Proteobacteria, Actinobacteria, Acidobacteria, Bacteroidetes and Chloroflexi were the dominant phyla in sorghum rhizosphere soils. Comparative analysis of samples showed that the bacterial composition of sorghum rhizosphere under salt stress was less affected by growth stage, and the bacterial community structure of sorghum rhizosphere changed with the aggravation of salt stress. Through WGCNA analysis of all filters OTUs, a total of 12 gene co-expression module were identified, pink module had significantly positive correlation with salinity and greenyellow module had significantly positive correlation with period and the contents of total phenols and flavonoids after salt treatment. The bacterial co-occurrence network was more complex in low salt stress soils than in higher salt stress soils, reflected by more nodes and more edges. The 13 network key OTUs were identified, OTU8480, OTU6866, OTU3247 and OTU3499 was the keyOTUs in S0 network; OTU6895, OTU4206, OTU6470, OTU1810 and OTU4916 in S3 network; OTU4217, OTU8426, OTU4847, and OTU6066 were the key OTUs in S7 network. 【Conclusion】The composition of rhizosphere bacteria in both varieties of sorghum has been modified in response to salt stress. After salt stress, with more complex co-occurrence network and tighter connections between OTUs of rhizosphere soil bacterial communities.

Key words: sorghum, rhizosphere, microbial community structure, salt stress

高玉坤, 张建东, 杨溥原, 陈东明, 王志博, 田颐瑾, Zakey Eldinn.E.A.Khlid, 崔江慧, 常金华. 高粱根际土壤细菌群落对盐胁迫的响应[J]. 生物技术通报, 2024, 40(4): 203-216.

GAO Yu-kun, ZHANG Jian-dong, YANG Pu-yuan, CHEN Dong-ming, WANG Zhi-bo, TIAN Yi-jin, Zakey Eldinn. E. A. Khlid, CUI Jiang-hui, CHANG Jin-hua. Responses of Sorghum Rhizosphere Soil Bacterial Communities to Salt Stress[J]. Biotechnology Bulletin, 2024, 40(4): 203-216.

图/表 9

表1 盐胁迫对株高和根系TPC、TFC的影响

Table 1 Effect of salt stress on plant height, TPC and TFC of sorghum roots

指标Items	时期Period	S0	S3	S7
株高 PH/cm	拔节期 E	67.9±6.9a	75.8±3.8a	54.6±4.6b
	开花期 F	115.8±4.3b	135.7±4.2a	92.6±2.6c
	成熟期 M	164±4.6b	185.3±4.1a	136.5±3c
总酚TPC/（mg·100 g^-1）	拔节期 E	60.92±19.04b	243.92±6.03a	209.38±23.76a
	开花期 F	222.89±2.72c	315.09±5.19a	293.09±15.88b
	成熟期 M	240.44±53.08a	285.43±7.59a	250.61±7.81a
总黄酮TFC/（mg·100 g^-1）	拔节期 E	50.5±17.02b	180.46±20.69a	175.15±13.72a
总黄酮TFC/（mg·100 g^-1）	开花期 F	182.05±3.18c	342.78±62.02b	423.93±22.05a
	成熟期 M	208.04±8.17b	273.82±11.18a	220.24±18.35b

表2 根际微生物群落测序质量

Table 2 Sequencing quality of rhizosphere microbial community

样本 Sample	有效序列数目 Seq_num	碱基数 Base_num/bp	样本序列平均长度 Mean_length/nt	样本最短序列长度 Min_length/nt	样本最长序列长度 Max_length/nt
ES0	162 093	67 727 026	417.82	252.00	506.33
ES3	160 893	67 266 875	418.08	244.33	491.67
ES7	172 634	72 184 160	418.14	247.67	486.33
FS0	171 403	71 496 233	417.13	214.33	468.33
FS3	157 105	65 548 212	417.20	271.00	474.00
FS7	161 222	67 470 566	418.48	235.00	490.67
MS0	151 139	63 252 701	418.48	227.00	465.00
MS3	148 582	61 959 724	417.02	247.33	477.00
MS7	146 276	61 086 964	417.61	245.33	471.00

图1 不同处理高粱土壤细菌测序结果评估和α多样性分析 A：丰度等级曲线；B：稀释曲线；C和D：Shannon、Simpson、ACE和Chao1指数

Fig. 1 Evaluation of sequencing results and Alpha diversity analysis of soil bacterial in sorghums soil samples under different treatments A: Rank abundance curve; B: dilution curves; C and D Shannon, Simpson, ACE and Chao1 indices, respectively

图2 根际土壤样本分析 A：基于PCoA的样本Beta多样性分析；B：基于PLS-DA的样本分组分析

Fig. 2 Analysis of sorghum rhizosphere soil samples A: Sample Beta diversity analysis based on PCoA. B: Sample grouping analysis based on PLS-DA

图3 根际微生物群落结构 A：不同样品中OTU数目花瓣图；B：门和属水平各样品细菌群落结构图；C、D：根际土门和属水平组间差异显著水平，*、**、***分别表示处理间达0.05、0.01和0.001显著性水平

Fig. 3 Bacterial community structure of sorghum rhizosphere A: Flower plot analysis for bacterial species（OTU）of different samples. B: Map of bacterial community structure at the phylum and genus level. C and D indicate phylum and genus distribution among different groups of rhizosphere soils, respectively. *, **, and *** indicate significant differences at the 0.05, 0.01, and 0.001 levels, respectively

图4 软阈值的确定和模块划分 A: 纵坐标表示无尺度网络模型指数；B: 纵坐标表示每一个软阈值对应的网络平均连接度；A 和 B 的横坐标均表示软阈值。C: 使用动态剪切算法得到的OTU模块和合并后的模块

Fig. 4 Determination of soft threshold and module division The ordinate of A indicate the scale-free network model index; the ordinate of B indicate the average network connectivity corresponding to each soft threshold; the abscissas of A and B both indicate the soft threshold ; C：OTU modules obtained by dynamic shearing algorithm and after merging similar expression patterns

图5 高粱根际细菌WGCNA分析 A：各模块中的细菌门组成；B：OUTs共表达模块与性状的关联热图；C和D：Pink和 greenyellow 模块核心OTUs共表达网络

Fig. 5 Analysis of WGCNA in sorghum rhizosphere bacteria A: Bacterial phylum composition in each module. B: Association analysis of OTUs co-expression network modules with traits. C and D indicate the hub OTUs co-expression network in pink and greenyellow module, respectively

图6 系统发生树和16S细菌菌群功能预测 A ：盐胁迫高粱根际微生物的系统发生进化关系；B：盐胁迫高粱根际微生物的功能预测；C：基于PCA的样本COG功能预测分析

Fig. 6 A phylogenetic tree showing the relationship among salt-treated groups and microbial functional features in salt-treated soil groups via COG analysis A: Evolutionary relationship in rhizosphere soil in four salt-treated soil groups. B: The microbial functional features in three salt-treated soil groups sorghum COG analysis. C: Microbial functional features in salt-treated soil groups via COG analysis based on PCA

图7 高粱根际细菌共现网络分析 A: 高粱根际细菌OTUs之间的模块关联网络； B：不同盐胁迫下高粱根际细菌网络物种组成（门水平）；C:不同盐胁迫下高粱根际细菌网络拓扑参数； D：不同盐胁迫处理节点在模块中拓扑角色的节点分布

Fig. 7 Co-occurrence network in sorghum rhizosphere bacteria A: Network revealing the modular associations among sorghum rhizosphere bacterial Otus. B: Species composition of sorghum rhizosphere bacterial network under different salt stress(phylum level). C: Sorghum rhizosphere bacterial network topological characteristics under different salt stress treatments. D: Distribution of nodes based on their topological roles in modules under different salt stress treatments

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