【作者】 马晓彤；

【导师】 姜瑞波；

【作者基本信息】 中国农业科学院 ， 农业资源与利用， 2009， 硕士

【摘要】 苜蓿是一种具有较高经济价值和营养价值的多年生豆科牧草。种植苜蓿既可以为畜禽提供优质的绿色蛋白饲料,又可以改良土壤,提高肥力,对生态环境具有深远的意义。提高苜蓿产量有效的方法是给苜蓿接种与之相匹配的根瘤菌。本研究是以中国农业微生物菌种保藏管理中心国家农业菌库中7株推广应用的苜蓿根瘤菌与6个苜蓿品种进行的共生匹配的初步探讨,并对7株根瘤菌采用生理生化特性反应、Biolog细菌鉴定系统和16S rDNA序列分析方法进行系统鉴定。通过根瘤菌与不同苜蓿品种在无氮水培营养液中进行结瘤试验和有效性分析,初筛出与对照相比共生匹配效果较好根瘤菌6株,苜蓿品种5个,共15个组合。将这15个组合进行土壤盆栽试验,分析了5个苜蓿品种接种6株根瘤菌的共生固氮生长效应,筛选出每一个苜蓿品种最佳共生效应的根瘤菌菌株,初步确立苜蓿根瘤菌与苜蓿品种共生匹配的优良组合5个,将复筛后的优良组合进行田间效果试验,为苜蓿根瘤菌更好地推广应用奠定基础。试验结果如下:1.将7株根瘤菌与6个苜蓿品种通过滤纸桥法,在无氮水培营养液中进行苜蓿根瘤菌与苜蓿品种的共生匹配结瘤试验,比较各个匹配组合与对照的差异,初筛出与对照相比较结瘤效果较好的共生匹配组合15个。2.土壤盆栽试验模拟田间试验的土壤环境,采用土壤不灭菌进行根瘤菌与苜蓿品种的共生匹配试验,对初筛出的较好根瘤菌与苜蓿组合在土壤中的适应性、结瘤能力和固氮有效性进行研究。结果表明,不同苜蓿品种接种根瘤菌ACCC17674与对照相比在结瘤率、根瘤数量、植株高度、植株鲜重、植株干重和全氮上有显著提高,且与其它菌株间差异显著,说明ACCC17674具有较强广谱性。共筛出共生匹配优良组合5个,分别是阿尔冈金苜蓿与ACCC17674、金皇后苜蓿与ACCC17674、牧歌苜蓿与ACCC17674、淮阴苜蓿与ACCC17674和保定苜蓿与ACCC17674。3.将复筛出的5个优良组合进行田间小区试验,结果表明,不同苜蓿品种接种ACCC17674与对照相比结瘤率、植株高度、干重、分枝数、全氮和产草量显著提高。最终得到3个共生匹配的优良组合,进一步验证了ACCC17674是一株广谱菌株,可以进一步推广应用到实际生产。4.在七株供试根瘤菌的生理生化特性试验中,在耐酸碱性方面,菌株ACCC17674和ACCC17675耐酸碱的pH值生长范围在4.0-9.5之间,耐盐性可达4.5%左右,说明两株菌在田间应用时,能够适应土壤pH值的较强变化和耐受高盐土壤环境。5.利用Biolog细菌鉴定系统和16S rDNA序列分析对供试7株根瘤菌进行系统鉴定时,7株菌均属于苜蓿中华根瘤菌(Sinorhizobium melilot),其鉴定结果与传统鉴定方法的鉴定相一致。在传统生理生化鉴定方法的基础上,利用当代先进的分析鉴定系统对根瘤菌的鉴定更系统更全面。更多还原

【Abstract】 The alfalfa is one kind of perennial leguminous forage with high economic value and nutritional value. The alfalfa can not only provide the high quality green protein feed for the domesticated fowl, but also improve soil and enhance the fertility, which is profoundly significant to the ecological environment. The effective method of increasing the alfalfa yield is the vaccination of the matching nodule bacteria on it. In the present study, the symbiotic of 7 strains of alfalfa rhizobia from Agricultural Culture Collection of China (ACCC) and 6 alfalfa varieties were investigated preliminarily and 7 Sinorhizobium meliloti were identified systemically by the reaction characteristic of the physiological biochemistry, the Biolog system of bacterim identification and the 16S rDNA sequential analysis method.Through nodulation experiments and effectiveness analysis of nodule bacterium and different alfalfa varieties in the non-nitrogen liquid medium, compared with the control, 6 rhizobia and 5 alfalfa varieties with better symbiotic matching effects were screened and 15 combinations were developed. The pot trial was conducted with these 15 combinations and the symbiotic nitrogen fixation growth effects of 5 alfalfa varieties vaccinated with 6 rhizobia were studied, and the rhizobia with the best symbiotic effect on ach alfalfa variety was selected and 5 combinations of the alfalfa nodule bacteria and the alfalfa variety with good symbiotic matching were screened, followed by field trial of the 5 combinations, which will be the foundation. of promoting the application of alfalfa nodule bacteria.The results were as following:1. The symbiotic matching experiment between 7 rhizobia and 6 alfalfa varieties were conducted by filter paper bridge culture method, in the non-nitrogen liquid medium culture and the differences between each matching combination and the control were compared. 15 combinations with better effect of symbiotic matching were screened.2. By the simulation of the field soil environment in pot trial, the symbiotic matching test between rhizobia and the alfalfa varieties was conducted with antiseptic soil, and the adaptability of screened rhizobia and alfalfa combination, nodulation ability and the efficiency of nitrogen fixation were studied. The results indicated that nodulation rate, nodule quantity, the plant height, the plant fresh weight and dry weight, the total nitrogen when rhizobium ACCC17674 vaccinated on the different alfalfa varieties were significantly increased, compared with control and significant differenced between ACCC17674 and the other strains were found. It is explained that ACCC17674 is a broad-spectrum strain. Finally, 5 combinations were screening respectively Algonquin, Golden Empress, Madrigal, HuaiYin and Baoding with ACCC17674 .3. A field plot trial was conducted with 5 screened good combinations. The results showed that nodulation rate, the plant height, branch number, the total nitrogen and yield were significantly increased and 3 combinations with good symbiotic matching were obtained, which had the potential of application in practical production.It is explained that ACCC17674 is a broad-spectrum strain. 4. In the physiological biochemistry characteristics reaction test of 7 rhizobia, strains ACCC17674 and ACCC17675 could grow with the pH value of 4.0-9.5 and 4.5% salt, and it suggested that in field application, the 2 strains could be adapted to the soil of with high pH value change and high salt content, with the good adaptability to environment and the higher tolerance.5. 7 Sinorhizobium meliloti identified by Biolog identification system and the 16S rDNA sequence analysis, the results were consistent with the traditional methods. Therefore, the advcanced analysis methods were more systematic and comprehensive on the basis of traditional methods in bacterial identification.更多还原