七彩花生根瘤内生菌对氮添加的响应及其与植物化学计量特征的关系

doi:10.17521/cjpe.2024.0002

摘要/Abstract

摘要： 豆科植物根瘤是根瘤菌生物固氮的场所, 含有多种微生物, 即根瘤内生菌, 其对植物的生长和营养具有重要作用。但根瘤内生菌群落结构对氮的响应及其与植物生态化学计量特征之间的关系还有待研究。该研究以七彩花生(Arachis hypogaea ‘Qicai’)为实验材料, 设置3个施氮处理(N0: 0 kg·hm^-2, N1: 140 kg·hm^-2, N2: 280 kg·hm^-2)进行大田实验, 测定叶片碳(C)、氮(N)、磷(P)含量和根瘤内生菌群落结构及丰度变化, 并采用PICRUSt 2软件进行功能预测分析。结果表明: (1)根瘤内生菌种类丰富, 共含有来自546个科1 049个属的微生物。其中主要的结瘤共生菌——慢生根瘤菌属(Bradyrhizobium)丰度占比仅为27.83%, 其他微生物丰度的总占比超过70%, 其中伯克霍尔德菌属(Burkholderia-Caballeronia-Paraburkholderia)和肠杆菌属(Enterobacter)的平均丰度占比均超过了10%。(2)施氮降低了根瘤内生菌菌群多样性, 并改变了其结构组成, 其中部分来自慢生根瘤菌属、肠杆菌属、科萨克氏菌属(Kosakonia)、泛菌属(Pantoea)的微生物相对丰度显著增加, 而一些来自根瘤菌属(Allorhizobium-Neorhizobium-Pararhizobium-Rhizobium)、中慢生根瘤菌属(Mesorhizobium)、分枝杆菌属(Mycobacterium)、伯克霍尔德菌属的微生物相对丰度显著降低, 且部分属相对丰度与植物化学计量特征具有紧密的相关关系。(3)共现网络分析结果显示, 部分菌群模块与叶片C、N、P含量及化学计量特征具有显著相关关系; 同时, PICRUSt 2功能预测结果也显示部分微生物C、N、P代谢酶功能与七彩花生叶片C、N、P含量及化学计量特征具有显著相关关系, 表明菌群结构与植物养分含量及其平衡之间具有紧密关系。综上, 七彩花生根瘤内生菌种类丰富, 且菌群结构和功能对氮添加较为敏感, 未来研究中可通过微生物分离以及合成菌群技术进一步发掘氮耐受型和敏感型内生菌的功能。

关键词: 七彩花生, 施氮, 根瘤内生菌, 化学计量特征, PICRUSt 2

Abstract:

Aims The root nodules of leguminous plants are the site of biological nitrogen fixation by rhizobia, and nodules also harbor diverse endophytes, which play important roles in plant growth and nutrient status. However, how root nodule endophytes respond to nitrogen input and the associations of endophyte composition with plant ecological stoichiometric characteristics remain largely unkown.

Methods We planted Arachis hypogaea‘Qicai’ in a field experiment with three levels of nitrogen treatment (N0: 0 kg·hm^-2, N1: 140 kg·hm^-2, N2: 280 kg·hm^-2). We also quantified the structures of endophyte communities and the contents of carbon (C), nitrogen (N) and phosphorus (P) in leaves, then the endophytic functions were predicted by PICRUST 2 software.

Important findings The results showed that endophytes are extremely diverse, with a total of 546 families and 1 049 genera. Among them, Bradyrhizobium accounted for 27.83% in relative abundance, while other genera accounted for more than 70%. The average proportion of Burkkholderia-Caballeronia-Paraburkholderia and Enterobacter was more than 10%. Nitrogen application decreased the diversity of endophytes and shifted the community composition, and the relative abundance of microorganisms from Bradyrhizobium, Enterobacter, Kosakonia and Pantoea increased significantly, while those from Allorhizobium-Neorhizobium-Pararhizobium- Rhizobium, Mesorhizobium, Mycobacterium and Burkholderia-Caballeronia-Paraburkholderia were significantly decreased. The relative abundance of part of genera was closely correlated with the stoichiometric characteristics of the plants. The results of co-occurrence network analysis show that some modules are significantly correlated with the contents and stoichiometric characteristics of C, N, and P in the leaves. Meanwhile, the results of PICRUSt 2 functional prediction show that the functions of microbial C, N, and P metabolic enzymes are significantly correlated with the contents and stoichiometric characteristics of C, N, and P in leaves, indicating that there is a close association of the microbial community structure with the contents and balance of plant nutrients. In summary, our study shows that the composition of endophytes in the root nodules of the Arachis hypogaea‘Qicai’ is extremely diverse, and the community structures and functions are sensitive to nitrogen addition. In future studies, the functions of nitrogen-tolerant and sensitive endophytes can be further explored through microbial isolation and synthetic microbial community techniques.

Key words: Arachis hypogaea ‘Qicai’, nitrogen fertilization, root nodule endophyte, stoichiometric characteristics, PICRUSt 2

李林, 孙毅, 杨晓琼, 方海东, 闫帮国. 七彩花生根瘤内生菌对氮添加的响应及其与植物化学计量特征的关系. 植物生态学报, 2024, 48(10): 1374-1384. DOI: 10.17521/cjpe.2024.0002

LI Lin, SUN Yi, YANG Xiao-Qiong, FANG Hai-Dong, YAN Bang-Guo. Response of endophytes in root nodules of Arachis hypogaea ‘Qicai’ to nitrogen addition and its relationship with plant stoichiometry characteristics. Chinese Journal of Plant Ecology, 2024, 48(10): 1374-1384. DOI: 10.17521/cjpe.2024.0002

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链接本文: https://www.plant-ecology.com/CN/10.17521/cjpe.2024.0002

https://www.plant-ecology.com/CN/Y2024/V48/I10/1374

图/表 5

图1 不同施氮量下七彩花生叶片碳(C)、氮(N)、磷(P)含量及化学计量特征变化。相同小写字母表示处理间不存在显著差异(p > 0.05)。N0, 0 kg·hm-2; N1, 140 kg·hm-2; N2, 280 kg·hm-2。

Fig. 1 Changes of carbon (C), nitrogen (N) and phosphorus (P) contents and stoichiometric characteristics of Arachis hypogaea ‘Qicai’ leaves under different nitrogen addition treatments. The same lowercase letters indicate no significant differences between treatments (p > 0.05). N0, 0 kg·hm-2; N1, 140 kg·hm-2; N2, 280 kg·hm-2.

图2 不同施氮处理下七彩花生根瘤内生菌多样性(A)、主要内生菌各属丰度变化(B)、主坐标分析(PCoA) (C)、根瘤内生细菌丰度变化火山图(D)。A图中不同小写字母表示处理间差异显著(p < 0.05)。D图中的点表示一个扩增子序列变体, 红色表示在氮添加下显著上调, 绿色表示显著下调。N0, 0 kg·hm-2; N1, 140 kg·hm-2; N2, 280 kg·hm-2。Allorhizobium-Neorhizobium- Pararhizobium-Rhizobium, 根瘤菌属; Bradyrhizobium, 慢生根瘤菌属; Burkholderia-Caballeronia-Paraburkholderia, 伯克霍尔德菌属; Enterobacter, 肠杆菌属; Kosakonia, 科萨克氏菌属; Mycobacterium, 分歧杆菌属; Novosphingobium, 新鞘氨醇菌属; Pantoea, 泛菌属; Saccharimonadales, 糖单孢菌; Unassigned, 未分类。*, p < 0.05。

Fig. 2 Diversity (A), changes in the relative abundance of dominant genera (B), principal coordination analysis (PCoA) (C) and volcano plot illustrating changes in the relative abundance (D) of endophytes in root nodules under different nitrogen addition treatments of Arachis hypogaea ‘Qicai’. Different lowercase letters in A indicate significant differences among treatments (p < 0.05). Each point represents an amplicon sequence variant (ASV) in D. Each red point indicate an enriched ASV while each blue point represents a depleted ASV. N0, 0 kg·hm-2; N1, 140 kg·hm-2; N2, 280 kg·hm-2. *, p < 0.05.

表1 七彩花生根瘤内生细菌主要属相对丰度与叶片碳(C)、氮(N)、磷(P)含量及化学计量特征的关系

Table 1 Relationship between relative abundance of Arachis hypogaea ‘Qicai’ root nodule endophytes and leaf carbon (C), nitrogen (N) and phosphorus (P) stoichiometric characteristics

相关性系数 Correlation coeffficient	C	N	P	C:N	C:P	N:P
根瘤菌属 Allorhizobium-Neorhizobium- Pararhizobium-Rhizobium	0.00	-0.35^*	-0.11	0.37^**	0.13	-0.12
慢生根瘤菌属 Bradyrhizobium	0.01	0.17	0.27	-0.19	-0.23	-0.08
伯克霍尔德菌属 Burkholderia-Caballeronia- Paraburkholderia	0.37^**	0.16	-0.03	-0.08	0.06	0.10
肠杆菌属 Enterobacter	-0.22	0.18	-0.05	-0.23	0.02	0.18
科萨克氏菌属 Kosakonia	-0.27	-0.09	-0.04	0.03	-0.01	-0.03
分枝杆菌属 Mycobacterium	0.03	-0.16	0.13	0.18	-0.11	-0.20
新鞘氨醇菌属 Novosphingobium	0.18	-0.21	-0.04	0.24	0.09	-0.09
泛菌属 Pantoea	0.08	-0.02	-0.04	0.04	0.04	0.01
糖单孢菌 Saccharimonadales	-0.04	-0.21	0.03	0.17	-0.03	-0.15

图3 PICRUSt 2预测根瘤内生菌酶功能对施氮量的响应及其与叶片化学计量特征的关系。A, 酶活性变化(平均值±标准差)。B, 酶活性主坐标分析(PCoA)。C, 酶活性与叶片化学计量特征的关系。N0, 0 kg·hm-2; N1, 140 kg·hm-2; N2, 280 kg·hm-2。C, 碳; N, 氮; P, 磷。3-Phytase, 3-植酸酶; 4-Phytase, 4-植酸酶; Acid phosphatase, 酸性磷酸酶; Alkaline phosphatase, 碱性磷酸酶; Alpha-amylase, α-淀粉酶; Alpha-glucosidase, α-糖苷酶; Ammonia monooxygenase, 氨单加氧酶; Beta-glucosidase, β-糖苷酶; Cellulase, 纤维素酶; Chitinase, 几丁质酶; Nitrate reductase, 硝酸还原酶; Nitric oxide dioxygenase, 一氧化氮双加氧酶; Nitrite reductase (NADH), 亚硝酸还原酶(NADH); Nitrite reductase (NO-forming), 亚硝酸盐还原酶(NO-生成); Nitronate monooxygenase, 硝基单加氧酶; Nitrous-oxide reductase, 一氧化二氮还原酶; Nitrogenase, 固氮酶。*, p ≤ 0.05; **, p ≤ 0.01。A中不同小写字母表示不同处理间差异显著(p < 0.05)。

Fig. 3 Response of root nodule endophyte enzyme functions (predicted by PICRUSt 2) to nitrogen addition treatments and their association with leaf stoichiometric characteristics. A, Changes in enzyme activities (mean ± SE). B, Principal component analysis (PCoA) of enzyme activity. C, Relationships between enzyme activities and leaf stoichiometric characteristics. N0, 0 kg·hm-2; N1, 140 kg·hm-2; N2, 280 kg·hm-2. C, carbon; N, nitrogen; P, phosphorus。*, p ≤ 0.05; **, p ≤ 0.01. Different lowercase letters in A indicate significant differences among treatments (p < 0.05).

图4 七彩花生根瘤内生菌群落共现网络分析。A, 根瘤细菌群共现网络。B, 不同菌群模块丰度与叶片化学计量特征关系。C, 各模块主要菌属组成。N0, 0 kg·hm-2; N1, 140 kg·hm-2; N2, 280 kg·hm-2。C, 碳; N, 氮; P, 磷。Allorhizobium-Neorhizobium-Pararhizobium-Rhizobium, 根瘤菌属; Bradyrhizobium, 慢生根瘤菌属; Burkholderia- Caballeronia-Paraburkholderia, 伯克霍尔德菌属; Enterobacter, 肠杆菌属; Kosakonia, 科萨克氏菌属; Mycobacterium, 分歧杆菌属; Novosphingobium, 新鞘氨醇菌属; Pantoea, 泛菌属; Saccharimonadales, 糖单孢菌; Unassigned, 未分类。*, p ≤ 0.05; **, p ≤ 0.01。

Fig. 4 Co-occurrence network analysis of endophytic communities in root nodules of Arachis hypogaea ‘Qicai’. A, Cooccurrence network of root nodule endophytes. B, Associations between the relative abundance of different clusters with leaf stoichiometric characteristics. C, Main bacterial genus composition of each module. N0, 0 kg·hm-2; N1, 140 kg·hm-2; N2, 280 kg·hm-2. C, carbon; N, nitrogen; P, phosphorus. *, p ≤ 0.05; **, p ≤ 0.01.

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