在突尼斯北部和中部收集的23个土壤样品中,有16个有结节的Pisumsativum植物显示存在天然根瘤菌。总共选择了130个细菌菌株,并在PCR-RFLP分析后发现了三种不同的核糖型。rrs和四个管家基因的序列分析(recA,atpD,dnaK和glnII)将35个分离株分配给根瘤菌,R.ruizarguesonis,土壤杆菌,Ensifermeliloti和两个推定的基因组。拉格雷科是结瘤的最主要物种,占63%。分离株21PS7和21PS15被分配给R.ruizarguesonis,这是该物种在突尼斯的第一份报告。确定了两个推定的新谱系,由于菌株25PS6,10PS4和12PS15明显地与已知的根瘤菌物种聚集在一起,但在豆科动物复合体(Rlc)中,最密切相关的物种是R.indicum,具有96.4%的序列同一性。同样,菌株16PS2、3PS9和3PS18与苦参和拉格雷科表现出97.4%和97.6%的相似性,分别。基于16S-23S基因间间隔区(IGS)指纹,菌株与其地理位置之间没有明显的关联.根据nodC和nodA系统发育,Rlc物种的菌株,有趣的是,菌株8PS18被鉴定为E.meliloti,具有共生菌的共生基因,并聚集在两个不同的进化枝中,显示出共生体内的异质性。所有这些菌株都与豌豆植物结瘤和固定氮。然而,属于A.radiobacter的菌株和剩下的两个E.meliloti菌株无法结瘤。表明它们是非共生菌株。这项研究的结果进一步表明,突尼斯根瘤菌群落比以前报道的更加多样化。
Nodulated Pisum sativum plants showed the presence of native rhizobia in 16 out of 23 soil samples collected especially in northern and central Tunisia. A total of 130 bacterial strains were selected and three different ribotypes were revealed after PCR-RFLP analysis. Sequence analyses of rrs and four housekeeping genes (recA, atpD, dnaK and glnII) assigned 35 isolates to
Rhizobium laguerreae, R. ruizarguesonis, Agrobacterium radiobacter, Ensifer meliloti and two putative genospecies. R. laguerreae was the most dominant species nodulating P. sativum with 63%. The isolates 21PS7 and 21PS15 were assigned to R. ruizarguesonis, and this is the first report of this species in Tunisia. Two putative new lineages were identified, since strains 25PS6, 10PS4 and 12PS15 clustered distinctly from known rhizobia species but within the R. leguminosarum complex (Rlc) with the most closely related species being R. indicum with 96.4% sequence identity. Similarly, strains 16PS2, 3PS9 and 3PS18 showed 97.4% and 97.6% similarity with R. sophorae and R. laguerreae, respectively. Based on 16S-23S intergenic spacer (IGS) fingerprinting, there was no clear association between the strains and their geographic locations. According to nodC and nodA phylogenies, strains of Rlc species and, interestingly, strain 8PS18 identified as E. meliloti, harbored the symbiotic genes of symbiovar viciae and clustered in two different clades showing heterogeneity within the symbiovar. All these strains nodulated and fixed nitrogen with pea plants. However, the strains belonging to A. radiobacter and the two remaining strains of E. meliloti were unable to nodulate P. sativum, suggesting that they were non-symbiotic strains. The results of this
study further suggest that the Tunisian
Rhizobium community is more diverse than previously reported.