紫花苜蓿内生和非内生根瘤菌多样性及共生差异

Diversity and symbiotic difference of endophytic and non-endophytic rhizobia of alfalfa (Medicago sativa L.)

摘要:

【背景】对根瘤菌多样性的研究有助于推进根瘤菌种质资源的利用。【目的】研究紫花苜蓿内生和非内生根瘤菌的表型和遗传多样性，比较菌株在5个苜蓿品种上的共生效应，验证根瘤菌群体共生效应由苜蓿品种决定的假设。【方法】从甘肃省白银会宁旱作区、兰州安宁灌区、武威凉州灌区3个栽培区域的陇中、清水、WL168HQ、甘农3号、甘农9号等紫花苜蓿品种中分离内生(植株种子、花、叶、茎、根表皮、根中柱和根瘤)和非内生(根际土壤和田间土壤)根瘤菌菌株，通过表型数值分类、16S rRNA基因限制性片段长度多态性(Restriction fragment length polymorphism，RFLP)、16S rRNA基因测序、持家基因多位点序列分型(Multilocus sequence typing，MLST)，以及结瘤基因nodC和固氮基因nifH片段序列测定，研究紫花苜蓿根瘤菌的表型和遗传多样性，并采用主成分分析研究根瘤菌菌株在5个紫花苜蓿品种上的共生效应差异。【结果】共分离得到43株内生根瘤菌和10株非内生根瘤菌，叶片和花中没有分离到根瘤菌菌株。53株根瘤菌以及对照菌株R.GN5和S.12531表型特征数值分类聚为8个群，菌株表型多样性丰富。经16S rRNA-RFLP分析共形成22种RFLP分型组合，基因型Ⅰ分布最广泛(24)，其次为基因型Ⅻ (5)、ⅩⅤ (5)和ⅩⅨ (3)，其余16株菌各代表1种基因型，菌株遗传多样性丰富。16S rRNA基因测序和MLST分析将所有菌株划分为Rhizobium radiobacter、R. rosettiformans和Ensifer meliloti。仅从7株E. meliloti代表菌株和对照菌株S.12531中扩增到nodC和nifH基因，说明E. meliloti菌株均能结瘤固氮。E. meliloti菌株G3L3接种甘农3号，LP3、LL1和LL2接种陇中，QL2接种清水，LL1、LL2和WLP2接种WL169HQ苜蓿均能显著促进植株的单株结瘤数、地上干重和粗蛋白含量。E. meliloti菌株接种甘农3号、甘农9号和清水苜蓿品种后所有参数值在PC1轴上分别聚在?1?1之间，在PC3轴上聚在?1.5?1.5之间；接种陇中和WL168HQ苜蓿的参数值较分散，PC1轴上分散在?1.5?4之间，PC3轴上分散在?3?4之间。【结论】紫花苜蓿内生和非内生根瘤菌菌株多样性丰富，表型和遗传多样性与其来源没有直接关系。菌株G3L3与甘农3号，LP3、LL1、LL2与陇中，QL2与清水、LL1、LL2、WLP2与WL169HQ苜蓿品种共生匹配和适应能力强。在甘农3号、甘农9号和清水紫花苜蓿品种上群体共生效应相似，在陇中和WL168HQ紫花苜蓿上共生效应差异明显。本研究内生和非内生根瘤菌菌株的群体共生效应根据苜蓿品种而定，根瘤菌菌株与苜蓿品种间的信号识别程度存在差异。

Abstract:

[Background] Research on rhizobium diversity has paved the way for utilization of rhizobial germplasm resources. [Objective] To research the phenotypic and genetic diversity of endophytic and non-endophytic rhizobia of alfalfa (Medicago sativa L.), and verify the hypothesis that rhizobial symbiotic efficiency differed according to alfalfa variety by comparing their symbiotic difference on five alfalfa varieties. [Methods] Endophytic (seed, flower, leaf, stem, root epidermis, root stele, nodule) and non-endophytic (rhizosphere soil and field soil) bacteria isolates were collected from M. sativa cvs. Longzhong and Qingshui in arid crop area of Huining, Baiyin, M. sativa cv. WL168HQ in irrigated area of Anning, Lanzhou, and M. sativa cvs. Gannong No. 3 and Gannong No. 9 in irrigated area of Liangzhou, Wuwei, Gansu. Numerical analysis, 16S rRNA restriction fragment length polymorphism fingerprinting (RFLP), 16S rRNA gene sequencing, multilocus sequence typing (MLST) of concatenated sequences of atpD, glnII, and recA genes, and sequence analysis of symbiotic genes nodC and nifH were applied to study the phenotypic and genetic diversity of endophytic and non-endophytic rhizobia. A principal component analysis (PCA) was used to investigate their symbiotic differences on five alfalfa varieties as well. [Results] Totally 43 endophytes and 10 non-endophytic isolates were obtained. None were collected from flowers and leaves. The phenotypic diversity of these 53 isolates along with two reference strains (R.GN5 and S.12531) were abundant, with eight phenotypic clusters formed. Twenty-two RFLP patterns were produced after 16S rRNA-RFLP analysis, and the most widespread genotype among the isolates was that designated as genotype Ⅰ (24). Three other genotypes (Ⅻ, ⅩⅤ and ⅩⅨ) occurred less frequently in alfalfa symbionts (five, five and three). There were 16 genotypes specific to a single M. sativa isolate. According to the phylogenetic analyses of 16S rRNA gene and MLST, isolates were further classified into Rhizobium radiobacter, R. rosettiformans, and Ensifer meliloti. The nodC and nifH gene fragments were only amplified and sequenced from seven representative E. meliloti strains and reference strain S.12531, indicating that they were capable of nodulating alfalfa. The nodule number per plant, shoot dry weight and crude protein content of M. sativa cvs. Gannong No. 3 (inoculated with G3L3), Longzhong (inoculated with LP3, LL1 and LL2), Qingshui (inoculated with QL2), and WL168HQ (inoculated with LL1, LL2 and WLP2) were promoted simultaneously. The parameter values of M. sativa cvs. Gannong No. 3, Gannong No. 9, and Qingshui plants inoculated with the E. meliloti isolates clustered together, which ranged from ?1 to 1 in PC1 axis and ?1.5 to 1.5 in PC3 axis. Compared with these three alfalfa varieties, that of M. sativa cvs. Longzhong and WL168HQ plants dispersed greatly and ranged from ?1.5 to 4 in PC1 axis and ?3 to 4 in PC3 axis. [Conclusion] The phenotypic and genetic diversity of endophytic and non-endophytic rhizobia were abundant, and there was no direct relationship between diversity and strains’ origins. Strong mutualistic symbiosis and adaptability were presented between G3L3 and M. sativa cv. Gannong No. 3, LP3, LL1, LL2 and M. sativa cv. Longzhong, QL2 and M. sativa cv. Qingshui, and LL1, LL2, WLP2 and M. sativa cv. WL168HQ. The tested strains exhibited similar symbiotic efficiency when inoculated onto M. sativa cvs. Gannong No. 3, Gannong No. 9, and Qingshui plants, while an obvious symbiotic difference of rhizobial strains was observed in M. sativa cvs. Longzhong and WL168HQ plants. Their symbiotic efficiency varied according to alfalfa varieties, which manifested that the sensitivity of different alfalfa varieties to rhizobial strains may differ.