西北地区金属尾矿地根瘤菌的重金属抗性及其系统发育研究
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摘要
从陕西、甘肃金属尾矿废弃地采集鸡眼草、刺槐、胡枝子、截叶铁扫帚、草木樨、截叶铁扫帚、狼牙刺等19种豆科植物的根瘤,分离纯化得到188株根瘤菌。采用平板筛选的方法分析供试菌株对Cu~(2+)、Zn~(2+)、Pb~(2+)、Cd~(2+)、Cr6+、Hg~(2+)、Ni~(2+) 7种重金属的耐受能力、确定其最大抗性水平(MRL),并对其中9株对各种重金属均具有较好抗性的菌株进行系统发育地位研究。本实验首次对金属尾矿废弃地根瘤菌的重金属抗性及其系统发育地位进行了研究。
筛选与最大抗性水平分析结果表明:液体培养基比固体培养基中有较高的耐受性;不同菌株对重金属的耐受性存在较大差异,其中3株可同时耐受6种重金属,大部分菌株表现出对(<0.5 mmol/L)Hg~(2+)、Cd~(2+)、Cr6+敏感,而对(<2.5 mmol/L)Pb~(2+)不敏感。CCNWSX0403和CCNWSX0360均可耐受4.0 mmol/L Zn~(2+),分别可耐受2.4 mmol/L和2.8 mmol/L Cu~(2+),CCNWGS0139可耐受0.4 mmol/L Hg~(2+),CCNWSX0003可耐受2.4 mmol/L Ni~(2+),CCNWGS0284和CCNWGS0142可耐受4.8 mmol/L Pb~(2+)。CCNWSX0403的生理生化特性研究表明:供试菌株产碱,在4℃低温条件下不能生长,能在60℃高温瞬间处理10min和pH 5-12之间良好的生长;能够耐受5%的NaCl,耐受300μg /mL氨卞西林钠、50μg /mL硫酸链霉素、300μg /mL林可霉素、300μg /mL磷雷素钠、50μg /mL硫酸阿米卡星、150μg /mL青霉素钠、50μg /mL氯霉素、0.2% NaNO2、900 mg/L的苯酚。
对9株抗性菌株的16S rDNA全序列分析表明,CCNWGS0122、CCNWSX0003分别属于中慢生根瘤菌属(Mesorhizobium)和中华根瘤菌属(Sinorhizobium)。另外发现对Pb~(2+)、Hg~(2+)耐受性较好的4株菌CCNWSX0386、CCNWGS0139、CCNWGS0284和CCNWGS0142在分类地位上均属于土壤杆菌属(Agrobacterium);而属于慢生根瘤菌属(Bradyrhizobium)的3株菌CCNWGS0309、CCNWSX0403和CCNWSX0360对Cu~(2+)、Zn~(2+)耐受性较好。总体上来看, Agrobacterium对Hg~(2+)和Pb~(2+)的耐受性较好,Bradyrhizobium比Rhizobium、Sinorhizobium、Mesorhizobium有较强的重金属耐受性。
实验对重金属处理下抗性菌株与豆科植物的结瘤情况进行了研究。结果表明:低浓度的Cu~(2+)、Zn~(2+)促进根瘤菌与豆科植物结瘤,高浓度抑制结瘤,与对照相比高浓度重金属处理下根瘤菌结瘤的时间延迟,根瘤变小,相同浓度下Cu~(2+)对结瘤的影响大于Zn~(2+),重金属Cu~(2+)、Zn~(2+)对植物的地上部分和根部的影响同样表现为低浓度促进生长高浓度抑制生长,但差异不明显。Cu~(2+)对植物生长的影响大于Zn~(2+)的影响。
To screen heavy metals resistant rhizobial strains and study maximum resistance level (MRL), 188 rhizobial strains were isolated from 19 species of legume plants nodules: R.pseudoacacia, A.abrachystach, L. cuneata, S. viciifolia, L.bicolor Turcz., M. officinalis and so on, which generated in metal mining tailing in Shaanxi and Gansu province. These strains were used to screen resistance strains and analyses MRL to seven heavy metals: Cu~(2+)、Zn~(2+)、Pb~(2+)、Cd~(2+)、Cr6+、Hg~(2+)、Ni~(2+). We also studied the phylogeny of 9 resistance strains.This study reaserch the rhizobium resistance in the metal tailing and phylogeny of resistant strains for the first time.
The result showed that there were great differences in the resistance of rhizobial strains to heavy metals. The resistant strains have higher tolerance in liquid media (TY) than in soild (YMA). Strain CCNWSX0386, CCNWGS0138 and CCNWGS0310 was almost tolerant to six heavy metals tested. In general, all of the strains were senstive to low concentration (<0.4 mmol/L) Hg~(2+), Cd~(2+) and Cr6+. But most of strains were less sensitive to Pb~(2+) (<2.5 mmol/L). Strain CCNWSX0403 and CCNWSX0360 both tolerated Zn~(2+) 4.0 mmol/L and tolerated Cu~(2+) concentration up to 2.4mmol/L and 2.8mmol/L, respectively. Strain CCNWGS0139 could tolerate 0.4 mmol/L Hg~(2+),CCNWSX0003 could tolerate 2.4 mmol/L Ni~(2+) and CCNWGS0284, CCNWGS0142 could tolerate 4.8 mmol/L Pb~(2+).
One heavy metals resistant excellent strain CCNWSX0403 were characterized with physiological and biochemical characteristics. The result indicated that CCNWSX0403 can produce alkali. It has well growth on the plate with initial pH 5-12 and grow well after treated with 60℃for 10 min, but can not grow at 4℃incubate condition. The strain can tolerated 5% NaCl, 300μg/mL Ampicillin Sodium, 50μg/mL Streptomycin Sulfate, 300μg/mL Lincomycin, 300μg/mL Fosfomycin Sodium, 50μg/mL Amikacin Sulfate, 150μg/mL Benzylpenicillin Sodium, 50μg /mL Chloramphenicol, 0.2% NaNO2 and 900mg/L phenol.
16S rDNA sequences of nine resistant strains screened were analyzed. Strain CCNWGS0122 and CCNWSX0003 were identified as Mesorhizobium and Sinorhizobium, respectively. Four of strains, specifically CCNWSX0386, CCNWGS0139, CCNWGS0284 and CCNWGS0142, which had better resistance to Pb~(2+) and Hg~(2+) belonged to the genus of Agrobacterium. But three of strains, specifically CCNWGS0309, CCNWSX0403 and CCNWSX0360, which belonged to the genus of Bradyrhizobium had better resistance to Cu~(2+) and Zn~(2+). In general, Agrobacterium were highly resistant to Hg~(2+) and Pb~(2+), Bradyrhizobium had higher resistance to heavy metals than Rhizobium, Sinorhizobium and Mesorhizobium.
Experiment were performed to analyse the nodulation in the different Cu~(2+), Zn~(2+) concentration .The result indicate that low concentration of Cu~(2+), Zn~(2+) both can facilitate the nodulation and the high concerntration inhibit the nodulation. In contrast with control, the root nodule change smaller and the time of nodulation are delayed in the high concerntration heavy metal.The toxicity of Cu~(2+) are stronger than Zn~(2+) in the same concentration. The toxicity to the growth of plant has the same effect, but the effect is not significant. The phytoxicity effect of Cu~(2+) is stronger than Zn~(2+) to pant, which similar to the nodulation.
筛选与最大抗性水平分析结果表明:液体培养基比固体培养基中有较高的耐受性;不同菌株对重金属的耐受性存在较大差异,其中3株可同时耐受6种重金属,大部分菌株表现出对(<0.5 mmol/L)Hg~(2+)、Cd~(2+)、Cr6+敏感,而对(<2.5 mmol/L)Pb~(2+)不敏感。CCNWSX0403和CCNWSX0360均可耐受4.0 mmol/L Zn~(2+),分别可耐受2.4 mmol/L和2.8 mmol/L Cu~(2+),CCNWGS0139可耐受0.4 mmol/L Hg~(2+),CCNWSX0003可耐受2.4 mmol/L Ni~(2+),CCNWGS0284和CCNWGS0142可耐受4.8 mmol/L Pb~(2+)。CCNWSX0403的生理生化特性研究表明:供试菌株产碱,在4℃低温条件下不能生长,能在60℃高温瞬间处理10min和pH 5-12之间良好的生长;能够耐受5%的NaCl,耐受300μg /mL氨卞西林钠、50μg /mL硫酸链霉素、300μg /mL林可霉素、300μg /mL磷雷素钠、50μg /mL硫酸阿米卡星、150μg /mL青霉素钠、50μg /mL氯霉素、0.2% NaNO2、900 mg/L的苯酚。
对9株抗性菌株的16S rDNA全序列分析表明,CCNWGS0122、CCNWSX0003分别属于中慢生根瘤菌属(Mesorhizobium)和中华根瘤菌属(Sinorhizobium)。另外发现对Pb~(2+)、Hg~(2+)耐受性较好的4株菌CCNWSX0386、CCNWGS0139、CCNWGS0284和CCNWGS0142在分类地位上均属于土壤杆菌属(Agrobacterium);而属于慢生根瘤菌属(Bradyrhizobium)的3株菌CCNWGS0309、CCNWSX0403和CCNWSX0360对Cu~(2+)、Zn~(2+)耐受性较好。总体上来看, Agrobacterium对Hg~(2+)和Pb~(2+)的耐受性较好,Bradyrhizobium比Rhizobium、Sinorhizobium、Mesorhizobium有较强的重金属耐受性。
实验对重金属处理下抗性菌株与豆科植物的结瘤情况进行了研究。结果表明:低浓度的Cu~(2+)、Zn~(2+)促进根瘤菌与豆科植物结瘤,高浓度抑制结瘤,与对照相比高浓度重金属处理下根瘤菌结瘤的时间延迟,根瘤变小,相同浓度下Cu~(2+)对结瘤的影响大于Zn~(2+),重金属Cu~(2+)、Zn~(2+)对植物的地上部分和根部的影响同样表现为低浓度促进生长高浓度抑制生长,但差异不明显。Cu~(2+)对植物生长的影响大于Zn~(2+)的影响。
To screen heavy metals resistant rhizobial strains and study maximum resistance level (MRL), 188 rhizobial strains were isolated from 19 species of legume plants nodules: R.pseudoacacia, A.abrachystach, L. cuneata, S. viciifolia, L.bicolor Turcz., M. officinalis and so on, which generated in metal mining tailing in Shaanxi and Gansu province. These strains were used to screen resistance strains and analyses MRL to seven heavy metals: Cu~(2+)、Zn~(2+)、Pb~(2+)、Cd~(2+)、Cr6+、Hg~(2+)、Ni~(2+). We also studied the phylogeny of 9 resistance strains.This study reaserch the rhizobium resistance in the metal tailing and phylogeny of resistant strains for the first time.
The result showed that there were great differences in the resistance of rhizobial strains to heavy metals. The resistant strains have higher tolerance in liquid media (TY) than in soild (YMA). Strain CCNWSX0386, CCNWGS0138 and CCNWGS0310 was almost tolerant to six heavy metals tested. In general, all of the strains were senstive to low concentration (<0.4 mmol/L) Hg~(2+), Cd~(2+) and Cr6+. But most of strains were less sensitive to Pb~(2+) (<2.5 mmol/L). Strain CCNWSX0403 and CCNWSX0360 both tolerated Zn~(2+) 4.0 mmol/L and tolerated Cu~(2+) concentration up to 2.4mmol/L and 2.8mmol/L, respectively. Strain CCNWGS0139 could tolerate 0.4 mmol/L Hg~(2+),CCNWSX0003 could tolerate 2.4 mmol/L Ni~(2+) and CCNWGS0284, CCNWGS0142 could tolerate 4.8 mmol/L Pb~(2+).
One heavy metals resistant excellent strain CCNWSX0403 were characterized with physiological and biochemical characteristics. The result indicated that CCNWSX0403 can produce alkali. It has well growth on the plate with initial pH 5-12 and grow well after treated with 60℃for 10 min, but can not grow at 4℃incubate condition. The strain can tolerated 5% NaCl, 300μg/mL Ampicillin Sodium, 50μg/mL Streptomycin Sulfate, 300μg/mL Lincomycin, 300μg/mL Fosfomycin Sodium, 50μg/mL Amikacin Sulfate, 150μg/mL Benzylpenicillin Sodium, 50μg /mL Chloramphenicol, 0.2% NaNO2 and 900mg/L phenol.
16S rDNA sequences of nine resistant strains screened were analyzed. Strain CCNWGS0122 and CCNWSX0003 were identified as Mesorhizobium and Sinorhizobium, respectively. Four of strains, specifically CCNWSX0386, CCNWGS0139, CCNWGS0284 and CCNWGS0142, which had better resistance to Pb~(2+) and Hg~(2+) belonged to the genus of Agrobacterium. But three of strains, specifically CCNWGS0309, CCNWSX0403 and CCNWSX0360, which belonged to the genus of Bradyrhizobium had better resistance to Cu~(2+) and Zn~(2+). In general, Agrobacterium were highly resistant to Hg~(2+) and Pb~(2+), Bradyrhizobium had higher resistance to heavy metals than Rhizobium, Sinorhizobium and Mesorhizobium.
Experiment were performed to analyse the nodulation in the different Cu~(2+), Zn~(2+) concentration .The result indicate that low concentration of Cu~(2+), Zn~(2+) both can facilitate the nodulation and the high concerntration inhibit the nodulation. In contrast with control, the root nodule change smaller and the time of nodulation are delayed in the high concerntration heavy metal.The toxicity of Cu~(2+) are stronger than Zn~(2+) in the same concentration. The toxicity to the growth of plant has the same effect, but the effect is not significant. The phytoxicity effect of Cu~(2+) is stronger than Zn~(2+) to pant, which similar to the nodulation.
引文
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