【作者】 韩玉竹；

【导师】 黄建国；

【作者基本信息】 西南大学 ， 微生物学， 2012， 博士

【摘要】 拉巴豆(Dolichos lablab L.)属于豆科蝶形花亚科菜豆族扁豆属,是国内外热带和亚热带广泛分布的优良高产牧草和蔬菜,它们的根瘤菌可同时侵染同族的豇豆。目前,我国有关研究甚少,提高拉巴豆的产量品质是亟待解决的科学和生产问题。论文作者首先自主分离了拉巴豆根瘤菌,研究它们的生物学特性,从中筛选出比较优良的菌株,接种在大面积种植的拉巴豆(润高)上,研究它们在不同土壤、施肥,以及丛枝菌根真菌与根瘤菌双接种条件下,对拉巴豆的生长发育、营养特征,以及产量品质的影响,为拉巴豆的科学种植,提高产量品质奠定了基础。主要研究结果如下：1拉巴豆根瘤菌生物学特性及优良菌株筛选本项研究从拉巴豆根瘤中分离获得了9株根瘤菌,具有耐盐、耐酸碱、耐高温、耐染料和抗生素等特性。它们可耐0～6％NaCl,在pH4.0～11.0生长。这些菌株适宜生长的环境温度为27～29℃,pH为6.0-8.0,盐浓度为0-0.12%。其中,根瘤菌菌株R1和R2-2的固氮酶活性、生长素分泌量、溶磷能力显著高于其它菌株,可能具有较强的固氮促生效应。此外,R2-3能溶解无机和有机磷,R2-6和R3-2可在pH11.0正常生长,R1、R2-4、R2-6在6％NaCl生长良好,且表现出较好的溶磷和抗盐碱的效应。2不同根瘤菌对拉巴豆的接种效应研究以高产优质牧草拉巴豆(品种：润高)为材料(下同),分别接种9株根瘤菌,观测宿主植物生长、生理和养分吸收等指标等变化。结果表明,9株根瘤菌都能形成根瘤,影响拉巴豆生长和养分吸收等。其中,根瘤菌R1号和R2-2的促进拉巴豆生长和提高植株养分含量的效果均最好,生物量比不接种者分别提高了71.05%和67.62%,氮、磷、钾、钙、镁含量平均含量依次提高了2.21%、0.28%、1.37%、1.33%、0.16%。此外,9株根瘤菌还不同程度地提高了拉巴豆叶片的硝酸还原酶活性、叶绿素含量和根系活力。3拉巴豆根瘤菌对不同土壤的响应试验以重庆市典型、具有代表性的灰棕紫泥、红棕紫泥、灰岩黄壤为供试土壤,接种促生根瘤菌R1,研究了拉巴豆的结瘤性能、生长情况、光合速率、营养品质、矿质养分等。结果表明,在微酸性和酸性灰棕紫泥土壤上,拉巴豆的生长、产量和品质最好,显著优于灰岩黄壤和红棕紫泥,其原因可能是拉巴豆起源于热带和亚热带,长期适应了酸性土壤。在微酸性灰棕紫泥(pH6.14)和灰岩黄壤(pH6.45)中,接种根瘤菌能形成根瘤,但在酸性灰棕紫泥(pH4.09)和碱性红棕紫泥(pH7.50)中,形成根瘤极少或不形成根瘤,说明土壤酸碱度过高过低抑制根瘤形成。接种根瘤菌形成根瘤之后,不同程度地改善了拉巴豆氮、磷营养,提高了净光合速率,促进了生长,增加了生物量,改善了品质。拉巴豆生物量,氮、磷吸收量,收后土壤有效氮、磷含量与根瘤重量呈显著正相关,故根瘤重量可反映根瘤活性。此外,在微酸性灰棕紫泥土壤中,拉巴豆的根瘤数量比灰岩黄壤少53.32%,但根瘤体积较大,根瘤重量为灰岩黄壤的1.66倍,接种效应总体上优于灰岩黄壤。所以,在重庆市的拉巴豆栽培过程中,选择微酸性和酸性灰棕紫泥,并接种根瘤菌有益于高产优质。4施氮量对拉巴豆生长及结瘤效应的影响设置N0、N8、N40、N2004种施氮量(Ni=mg/kg干土)处理,接种根瘤菌,研究了氮肥对拉巴豆的生长、结瘤、养分吸收及根际微生物数量的影响。结果表明,施用氮肥后,根瘤的数量和重重均显著高于空白对照；随着施氮量的增加,根瘤的数量和重量表现出先增后降的趋势,说明拉巴豆结瘤需要有一定的氮素营养,但浓度过高则产生抑制作用。但是,施氮量越大,拉巴豆生物量和氮磷钾吸收量越高,总生物量依次比不施氮肥增加了19.85%(N8)、47.60%(N40)、65.55%(N200)。此外,在高氮施用条件下,拉巴豆根际细菌和自生固氮菌数最多；在低氮条件下,根际真菌和放线菌数最多。拉巴豆结瘤与生长、硝酸还原酶活性、叶绿素含量、根系活力及养分吸收与细菌和自生固氮菌数呈显著正相关。在本试验中的高氮施肥条件下,拉巴豆依然保持较高的结瘤能力,并增加磷和钾吸收,提高生物量,所以拉巴豆需氮量较高,建议在拉巴豆栽培实践中适量施用氮肥很有必要。5有机无机肥配施和根瘤菌接种对拉巴豆光合、营养和土壤酶的影响科学施肥是植物高产优质重要手段。通过盆栽试验,研究施用化肥、有机肥,有机无机配合施用条件下,接种根瘤菌对拉巴豆结瘤、生长、生理、光合、饲料品质、养分吸收、土壤酶活性及根际微生物数量的影响。结果表明,F05M0.5(50%有机肥+50%无机肥)和F0.75M0.25(25%有机肥+75%无机肥)施肥处理的拉巴豆生长最好,F(纯施化肥)和F025M0.75(75%有机肥+25%无机肥)次之,M(纯施有机肥)较差,CK(不施肥)最差,F0.5M0.5和F0.75M0.25处理比F分别增产19.46%和16.49%。F和CK结瘤数最少,瘤重也最低,但根瘤数和重量随有机肥施用的比例增加而提高。施肥后拉巴豆叶片的硝酸还原酶活性、叶绿素含量及根系活力均高于对照。拉巴豆的光合特性指标均呈典型的“单峰”曲线,峰值在10：30左右,但施用有机肥处理的光合能力显著高于单施化肥与对照处理。拉巴豆的光合能力与生长、生理及根瘤形成四者之间均有显著正相关。此外,施用有机肥显著改善拉巴豆饲用品质,尤以F05M0.5和F0.75M0.25最佳。并且有机肥也显著提高了土壤的过氧化氢酶、蔗糖酶、脲酶的活性,以及根际微生物数量。相关分析表明,拉巴豆根瘤重与地上部生物量、氮、磷、钾吸收量呈显著正相关,相关系数依次为0.6758*、0.7269*、0.9759**、0.9488**(n=36),说明根瘤形成改善了拉巴豆的氮、磷、钾营养,促进了生长,提高了品质。在重庆市的拉巴豆栽培实践中,提倡接种根瘤菌和有机无机肥适量配施。6AM真菌对拉巴豆生长及养分吸收的影响研究4种丛枝菌根(Arbuscular mycorrhizal, AM)真菌(3株根内球囊霉Glomus intraradices Smith&Schenck,BEG-193、BEG-141、BEG-167,1株幼套球囊霉Glomus etunicatum Becker&Gerdemann,BEG-168)对拉巴豆生长、生理及养分吸收的影响。结果表明：供试AM真菌均可侵染拉巴豆根系,促进拉巴豆生长和营养吸收等。其中,BEG-193和BEG-167的菌根效应最强,侵染率、促进生长和提高植株养分含量的效果均最好,生物量比不接种分别提高了40.19%和40.65%；氮、磷、钾的平均养分吸收量依次比不接种提高了55.48%、86.94%、35.79%。此外,4株丛枝菌根还不同程度地提高了拉巴豆叶片的硝酸还原酶活性、叶绿素含量及根系活力。7根瘤菌与AM真菌双接种对拉巴豆生长及结瘤效应的影响单接R1或BEG-193,及两者混接的综合效应表明,混接的促生效果最好,优于单接BEG193和R1,不接菌的对照最差。接种后拉巴豆叶片的硝酸还原酶活性、叶绿素含量、根系活力以及生物量均显著高于对照(R1、BEG-193、混接的生物量分别比不接菌增加了21.30%、45.28%、64.65%)。R1、BEG-193、混接处理,氮的积累量分别比不接菌高40.66%、63.17%、96.70%；磷的积累量分别高38.11%、84.40%、142.17%；钾的积累量分别高20.24%、54.16%、69.14%。拉巴豆根际的细菌、真菌、放线菌、自生固氮菌的数量以混接处理的数量最多,单接处理次之,不接菌最少。更多还原

【Abstract】 Dolichos lablab L. is a legume forage or vegetable, belonging to genus Dolichos, Phaseolus, Papilionoideae of leguminous. It was widely distributed in tropical and subtropical regions because of its high-yield and good feeding quality. In current study we separated rhizobia from D. lablab and screened the excellent strains through biological characteristics of the rhizobia, investigated the influence of rhizobia on growth, development, nutritional characteristics, the yield and quality of D. lablab under the conditions of different soils, fertilization rates, and double inoculation of arbuscular mycorrhizal fungi and rhizobium. The results have laid a foundation for scientific cultivation and improving yield and quality of D. lablab. The main results are as follows:1Biological characteristic of the rhizobia from Dolichos lablab and better rhizobia strains selectedNine rhizobia strains were separated from nodules of D. lablab. The strains were salt-resistant, acid and alkali-resistant, dye-resistant and antibiotic-resistant. They could tolerate a salt condition of0to6%NaCl, and could live in a condition of pH4to11. However, the most suitable conditions for the rhizobia were:temperature27to29℃, pH6.0to8.0, concentration of NaCl0to0.12%. Among the nine strains, the nitrogenase activity, secretion of growth hormone and dissolving phosphorus of rhizobium bacteria R1and R2-2were significantly higher than those of other strains, indicating that R1and R2-2might have strong nitrogen-fixing effect and growth-promoting effects and could be developed as bio-fertilizer for D. lablab L. R2-3could dissolve inorganic and organic phosphorus, R3-2and R2-6could grow in pH11.0, R1、R2-4、R2-6could grow in6%NaCl, and they had the effects of phosphorus-dissolved and salt tolerance.2Inoculation effects of different rhizobia strainsNine rhizobia were separately inoculated to D. lablab (Rungao). The results showed that all nine rhizobia could form nodules with D. lablab and had an effect on its growth and nutrient uptake etc. Among the nine strains, nitrogen-fixing and growth-promoting effects of R1and R2-2were the best. Compare to control group (non-inoculated group), the biomass increased71.05%(R1) and67.62%(R2-2) and the average content of nitrogen, phosphorus, potassium, calcium, magnesium increased2.21%,0.28%,1.37%,1.33%,0.16%. The inoculation also improved nitrate reductase activity, chlorophyll content in leaves and root vigour of D. lablab at different degrees.3Response of Dolichos lablab’s rhizobia to different soilsThe influence of rhizobia inoculation on nodulation, growth, photosynthesis, nutrient quality and absorption by Dolichos lablab as well as soil nutrients were conducted in four typical and representative soils widely distributed in Chongqing, two grey brown purple soils with pH4.06and6.14, a red brown purple soil with pH7.50and a yellow limestone soil with pH6.45. The results showed that the growth, yield and quality of D. lablab in the two grey brown purple soils with pH4.06and6.14were better than those in the yellow limestone soil and alkaline red brown purple soil. Rhizobia inoculation could form nodules in the slight acidic grey brown purple soil(pH6.14) and yellow limestone soil (pH6.45), could form fewer nodules or no nodules in acidic grey brown purple soil (pH4.09) and alkaline red brown purple soil (pH7.50), which indicated that higher or lower pH value was unfavorable for nodule formation. Nodule formation could improve D. lablab’s nitrogen and phosphorus nutrition, net photosynthetic rate, promote the growth, increase biomass and quality to some extent. Positive correlations were obtained between biomass, nitrogen and phosphorus absorptions, and the soil nitrogen and phosphorus and nodule weight, indicating the nodule weight might reflect the nodule activity. The nodule number in the slight acidic grey brown purple soil was53.32%lower than that in the yellow limestone soil, while the size was lager and the nodule weight was1.66time to that in the yellow limestone soil, showing better inoculation. The selection of slight acidic and acidic grey brown purple soils and rhizobia inoculation could increase the yield and improve the quality of D. lablab in Chongqing,4The influence of nitrogen fertilizer on growth and nodulation effect of Dolichos lablabThe plants were seeded in pots with four nitrogen levels, No, N8, N40, N200(Ni=mg/kg dry soil). All pots were inoculated with Rl to investigate the influence of nitrogen fertilization on the growth, nodulation, nutrition absorption and rhizosphere microorganisms of D. lablab. The nodule number and weight were significantly higher for nitrogen fertilized plants than the control. The nodule number and weight increased first and then decreased with increasing nitrogen levels. This indicated that nodule formation of D. lablab required a certain amount of nitrogen. However, too much nitrogen supply might have an inhibitory effect. The biomass and NPK (nitrogen, phosphorus, potassium) uptake of D. lablab increased with increasing nitrogen levels. Compared with the control, total biomass for each nitrogen fertilizer level increased19.85%(N8),47.60%(N40) and65.55%(N200), respectively. The number of rhizosphere bacteria and azotobacter were the largest with high nitrogen level. Under low nitrogen level, the number of rhizosphere fungi and actinomycetes were the largest. Nodule weight positively correlated with nitrate reductase activity, chlorophyll content, root vigour, nutrient absorption and the number of rhizosphere bacteria and azotobacter. It was concluded that the growth of D. lablab demand more nitrogen. Under high nitrogen level, it could also maintain high nodule formation ability, improved nutrient absorption and biomass. Therefore, it is necessary to supply appropriate amount of nitrogen in D. lablab’s cultivation practice. 5Effects of chemical fertilizer combined with organic manure and rhizobium inoculation on photosynthesis, nutrient absorption and soil enzymes of Dolichos lablabFertilization is one of the most important factors affecting the growth and feeding quality of forage. Chemical fertilizer combined with organic manure (CK, F, F0.75M0.25, F0.5M0.5, F0.25M0.75, M) were used to investigate the influence of fertilizer treatments on nodulation, growth, physiology, photosynthesis, feeding quality, nutrient absorption by D. lablab as well as soil enzyme activity and the quantity of rhizosphere microorganisms. The results showed D. lablab had better growth, yield and quality with F0.5M0.5and F0.75M0.25, followed by F and F0.5M0.5, and M, CK was the worst. Compared with F, the yield of F0.5M0.5and F0.75M0.25increased19.46%and16.49%, respectively. Plants with F and CK formed fewer nodules (19～26No./pot) and lower nodule weight (0.58-1.56g/pot). However, nodule number and nodule weight increased with increased manure proportion. When the proportion of organic fertilizer increased from25%to100%, nodule number increased from54to109nodule/pot, nodule weight from3.13to3.90g/pot. Fertilization could improve the nitrate reductase activity, chlorophyll content in leaves and root vigour. The diurnal variation of photosynthetic indexs under different fertilization were basically the same. There had no obvious lunch break. The diurnal variation of net photosynthetic rate, transpiration rate, stomatal conductance, CO2use efficiency, light use efficiency appeared typical "single peak" and the curve peak was around10:30PM. Different fertilization influenced the photosynthetic capacity of D. Lablab. The net photosynthetic rate, transpiration rate, stomatal conductance, CO2use efficiency, energy use efficiency in F0.5M0.5were the highest, CK and F were lower. The photosynthetic capacity was significantly positively correlated with growth, physiology and nodule formation. Organic fertilizer could improve D. lablab’s contents of crude protein, crude fat, crude ash and nitrogen, phosphorus, potassium, and thus improve feeding quality. The ground biomass of F0.5M0.5and F0.75M0.25were the highest, of which the contents of protein, crude fat, crude ash, soluble sugar yield and nitrogen, phosphorus, potassium, calcium, magnesium absorption were also the highest. Compared with pure chemical fertilizer, organic manure could improve the soil enzyme (catalase, invertase, urease) activities. Correlation analysis showed shoot biomass (r2=0.6758), nitrogen (r2=0.7269), phosphorus (r2=0.9759) and potassium (r2=0.9488) absorption were positively correlated with nodule weight. Nodule formation could improve nitrogen, phosphorus and potassium nutrition, promote growth, increase biomass and quality to some extent. In cultivation practice in Chongqing, the selection of chemical fertilizer combined with organic manure and rhizobium inoculation could increase the yield and improve the quality of D. lablab.6Influence of AM fungi on Dolichos lablab’s growth and nutrient absorptionFour Arbuscular mycorrhizal fungi (AMF)(three were Glomus intraradices, another was Glomus etunicatum) were inoculated to investigate the influence of AMF on growth, physiology and nutrient absorption of D. lablab. The results showed that four AMF could infect D. lablab and promote it’s growth and nutrient absorption. The AMF BEG-193and BEG-167had the strongest mycorrhizal effect with highest mycorrhizal infection rate, growth rate and nutrient content. Compared with control, the biomass of D. lablab increased40.19%(BEG-193) and40.65%(BEG-167), respectively. Averagely, absorption of NPK (nitrogen, phosphorus, potassium) increased55.48%,86.94%and35.79%, respectively. Furthermore, nitrate reductase activity, chlorophyll content in leaves and root vigour of plants inoculated with AMF improved at different degrees.7Influence of co-inoculation of rhizobia and AM fungi on Dolichos lablab’s growth and nodulation effectThe growth and nodulation effect of plants inoculated with both R1or BEG-193were better than those with single inoculation. The nitrate reductase activity, chlorophyll content in leaves, root vigour, biomass of double inoculated plants were significantly higher than those of single inoculation and uninoculated control. Compared with control, the biomass of R1、BEG-193and double inoculation increased21.30%,45.28%and64.65%, respectively; nitrogen accumulation increased40.66%,63.17%and96.70%, respectively; phosphorus accumulation increased38.11%,84.40%and142.17%, respectively; potassium accumulation increased20.24%,54.16%and69.14%, respectively. The number of bacteria, fungi, actinomycete and azotobacter in D. lablab’s rhizosphere were larger with treatment of double inoculation than single inoculation and control.更多还原