镉污染水稻种子内生细菌的分离及其耐镉性和植物促生性研究

Isolation of endophytic bacteria from cadmium-contaminated rice seeds and their cadmium tolerance and plant growth-promoting traits

摘要:

【目的】植物内生细菌在促进植物生长和重金属污染土壤修复中发挥着重要作用。本研究对镉污染水稻种子内生细菌进行分离和功能鉴定,从而确定其镉耐受性、植物促生性以及对镉胁迫下水稻种子萌发和幼苗生长的影响。【方法】采用乙醇-次氯酸钠联合灭菌法对水稻种子进行表面灭菌,采用标准平板培养法对内生细菌进行分离、纯化;对菌株16S rRNA基因进行扩增和测序,鉴定菌株的分类学地位;运用微量稀释法、电感耦合等离子体质谱法、比色法测定内生细菌的镉耐受性、镉去除率和植物促生性;采用浸种法进行菌剂侵染,观察施加菌剂对水稻种子萌发、幼苗生长和镉积累的影响。【结果】从两个水稻品种的种子中共分离得到133株内生细菌,分属于12个属的24个种。在低镉品种728B中分离得到83个菌株,主要包括假单胞菌属(34.94%)、芽孢杆菌属(28.92%)和类芽孢杆菌属(10.84%)菌株。在高镉品种博B中分离得到50个菌株,主要包括泛菌属(40%)、短小杆菌属(22%)和微杆菌属(12%)菌株。对24个代表性菌株的镉耐受性进行测定,发现镉离子对菌株的最小抑制浓度介于80-2 560 μmol/L,其中芽孢杆菌HNR-4具有最高的镉耐受性。在含有5 mmol/L CdCl₂液体培养基中培养24 h,HNR-4对镉的去除率达到77.57%。对24个菌株的植物促生性测定发现,IAA合成量为0.78-40.12 μg/mL;铁载体产生量为1.46-752.74 mg/L;溶磷量为0-3.10 mg/L;ACC脱氨酶比活力为0-9.22 U/mg。施加芽孢杆菌HNR-4菌剂显著提高了镉胁迫下水稻种子萌发率和幼苗茎叶长,减小了镉离子由根部向地上部分的转移系数。【结论】尽管不同镉污染水稻品种的种子内生细菌组成存在一定差异,但这些内生细菌均有不同程度的耐镉性和植物促生性。对耐镉内生细菌的分离和鉴定,有助于进一步探讨重金属污染下内生细菌与植物互作机制以及微生物应用于农田重金属污染修复的潜力。

Abstract:

[Objective] Endophytes play an important role in the promotion of plant growth and the remediation of heavy metal-contaminated soil.In this study,we isolated the endophytic bacteria from cadmium-contaminated rice seeds and analyzed their cadmium tolerance,plant growth-promoting traits,and effects on rice seed germination and seedling growth under cadmium stress.[Methods] First,we used ethanol and sodium hypochlorite to sterilize the surface of rice seeds and then isolated the endophytic bacteria by standard dilution plating method.Second,we amplified and sequenced the 16S rRNA genes of the strains and identified their taxonomic positions.Third,we analyzed the cadmium tolerance,cadmium removal rate,and plant growth-promoting traits of the endophytic bacteria via microdilution method,inductively coupled plasma mass spectrometry (ICP-MS),and spectrophotometry,respectively.Finally,we infected the rice seeds with bacterial inoculants to observe their effects on seed germination,seedling growth,and cadmium accumulation.[Results] We isolated a total of 133 endophyte strains belonging to 24 species of 12 genera from two rice cultivars.Eighty-three strains were isolated from low-Cd-accumulating caltivar 728B,including Pseudomonas(34.94%),Bacillus(28.92%),and Paenibacillus(10.84%).Fifty strains were isolated from high-Cd-accumulating caltivar BB,including Pantoea(40%),Curtobacterium(22%),and Microbacterium(12%).The minimum inhibitory concentrations of cadmium against 24 representative strains were 80-2 560 μmol/L.Bacillus sp.HNR-4 showed the highest cadmium tolerance.The cadmium removal rate of HNR-4 reached 77.57% after the strain was cultured in the liquid medium containing 5 mmol/L Cd²⁺ for 24 h.Most of the 24 strains were capable of producing plant growth-promoting substances,such as IAA (0.78-40.12 μg/mL),siderophore (1.46-752.74 mg/L),soluble phosphate (0-3.10 mg/L),and ACC deaminase (0-9.22 U/mg).Under cadmium stress,HNR-4 inoculant significantly increased the germination rate and seedling shoot length,while reduced the transfer coefficient of cadmium from root to shoot.[Conclusion] Although the composition of seed endophytic bacteria varied in different rice cultivars,these bacteria all showed different levels of cadmium tolerance and plant growth-promoting activities.Isolation and identification of Cd-resistant endophytic bacteria may help investigate the interaction between endophytic bacteria and host plants exposed to heavy metal contamination,and explore the potential of microbial remediation in farmland.