豆科树种根瘤菌共生体系的研究——Ⅰ.结瘤观察、分离、回接和交叉接种

<正>对21个豆科树种,包括苏木亚科5个属、含羞草亚科6个属及蝶形花亚科5个属进行了结瘤观察。在田间苗圃及温室盆栽条件下,用未经灭菌的豆科树种根际土壤进行接种,其中只有12种结瘤;苏木亚科5个属的6种全不结瘤,南京常见豆科树种国槐也不结瘤。结瘤豆科树种的根瘤都经分离、纯化。根据对菌落培养性状及菌体形态特征的观察,可见全系典型的根瘤菌;对各原分离树种进行回接试验,进一步确定为各该树种根瘤菌。 9个不同树种根瘤菌经纯培养后,对一些树种进行了广泛的交叉接种试验。接种寄主除各豆科树种外,还包括绿豆、赤豆和豇豆。从所得结果看,大体上可将豆科树种根瘤菌归入豇豆族中。用分离所得的9个豆科树种根瘤菌纯培养的混合菌液在人为条件下接种,自然条件下未见结瘤的苏木亚科各树种和含羞草亚科的孔雀豆仍不结瘤。用2,4-D处理,则根部肿大畸形,甚至形成不规则形状的瘤体,但与真正的根瘤显然不同。观察的21个树种中有4种是首次报道,2种与过去报道的结瘤情况不同。     

我国豆科树种结瘤情况的补充资料

<正>在１９９０年编写我国豆科树种结瘤情况的资料基础上,近三年又在江苏、山东、安徽、福建、云南等省部分地区野外调查及育苗观察了４８属１２２种豆科树种结瘤情况,其中３６种是国内首次观察。     

豆科树种凝集素和根瘤菌胞外多糖结合反应与结瘤的关系

通过3种结瘤豆科树种合欢（Albizzia julibrissin）、马蹄针（Sophora davidii）、刺槐（Robinia pseudoacacia）和3种不结瘤豆科树种双决明（Cassia bicapsularis）、伞房决明（C．corymbosa）、黄槐决明（C．suffruticosa）的凝集素与13种根瘤菌胞外多糖的结合反应，在合欢、马蹄针和刺槐这3种树种中除合欢的凝集素不能与92-76菌株结合外，其余都能发生结合反应。而不结瘤的双决明、伞房决明和黄槐决明中也存在能与根瘤菌发生反应的凝集素，其中黄槐决明的凝集素可以与这13种根瘤菌株的胞外多糖都能发生凝集反应，但最终不能结瘤。因此豆科树种的凝集素与根瘤菌胞外多糖的结合可能仅是根瘤菌被吸附于豆科树种的根表，是它们之间建立共生体系的一个前提条件。     

2,4—D诱地高粱结瘤与根瘤菌（ORS—571）共生固氮研究

报道了以植物激素２，４－Ｄ（２，４－二氯苯氧惭酸）处理高粱，并接种ＯＲＳ－５７１根瘤菌，诱导高粱根系结瘤并与结瘤组织共生，表现出固氮活性。     

苏门答腊金合欢(豆科)共生根瘤研究Ⅰ形态发育和细胞结构观察

苏门答腊金合欢 Acacia glauca(L.)Moench 为热带山地豆科绿肥植物,六十年代初从印尼引进厦门地区试种,经过多年来栽培驯化,已能适应当地条件正常生长并结瘤固氮。1982年经黄维南等(1982)检验,发现其根瘤固氮活力高于厦门当地常见的几种豆科树种,如台湾相思(Acacia confusa Merr.)、有刺金合欢[A.farnesiana(L.)Willd]、     

2,4-D诱导高粱结瘤与根瘤菌(ORS-571)共生固氮研究

报道了以植物激素２，４－Ｄ（２，４－二氯苯氧乙酸）处理高粱（甜杂１号Ｓｏｒｇｈｕｍｖｕｌａ－ｇａｒｅ），并接种ＯＲＳ－５７１根瘤菌，诱导高粱根系结瘤并与结瘤组织共生，表现出固氮活性     

山西省土著大豆根瘤菌共生结瘤固氮特性研究

在山西土壤气候条件下，大豆主栽品种自然结瘤率达９４．６％，单株结瘤量为３７．１±１５．２个，日固氮量０．４４～１，５６ｍｇ，每公顷约固氮３０ｋｇ。单株固氮总活性高峰期随品种和栽培条件的不同有所变化，而单瘤固氮活性的高峰均在花期。土著根瘤茵的固氮强度与结瘤量无相关性，固氮量与单瘤固氮酶活则成显著正相关（ｒ＝０．７１Ｐ＜０．０５）。覆膜栽培和秸秆还田均具有良好的共生固氮生态效应。     

2，4－D诱导高梁结瘤与根瘤菌共生固氮的研究

本文以植物激素２．４－Ｄ（２．４－二氯苯氧乙酸）处理高梁（甜杂１号ｓｏｒｇｈｕｍｖｕｌｇａ－ｒｅ）（终浓度为３ｐｐｍ，并接种根瘤菌（ＯＲＳ５７１），诱导根瘤茵与高梁结瘤组织共生。自１９８９年以来，多次进行实验，结果表明这种方法可以诱导高梁根系结瘤并与结瘤组织共生，表现固氮活性。本研究实验的重复性和再现性都很好。高梁在移苗后，５～７天进行处理，效果较好，一般结瘤率都超过５０％，最高可达８０％。接种后４周瘤的直径约２～８ｍｍ，制片显微镜观察，瘤组织内有大量的根瘤菌菌体，说明根瘤菌可以进入高梁根系，并在瘤内繁殖。结瘤植株经气相色谱仪乙炔还原法测定，能表达一定程度的固氮活性。     

人工诱导小麦结瘤的研究

对人工诱导小麦结瘤进行了研究．研究表明，在一定的培养条件下，以２，４－Ｄ诱导，豇豆根瘤菌侵染，小麦根部能形成大量的瘤状结构，这种结构类似于豆科根瘤，不仅有大量的分生细胞，而且还有一定数量的根瘤菌．这些细菌主要位于胞间隙中．在我们的实验条件下，瘤状结构的形成对小麦生长有一定的抑制作用．     

接种方式对花榈木幼苗结瘤效应的影响

【目的】探讨珍贵用材树种花榈木幼苗接种根瘤菌的有效方法。【方法】设计了浸种、浸根、拌种和浇灌4种接种方式进行盆栽试验,以不接根瘤菌的盆栽苗为对照,观察接种后幼苗根系形态、结瘤数量变化,并测定幼苗生长、生理生化指标及固氮相关指标。【结果】不同接种方式处理下花榈木幼苗根系松软、呈土黄色,幼瘤多呈球形、黄褐色,结瘤部位多位于侧须根系上。不同接种方式处理的花榈木幼苗结瘤数量和质量差异明显,浇灌处理的结瘤数量最多,鲜瘤生物量最大,结瘤率最高,其次是浸根处理。接种上根瘤的花榈木幼苗生长指标均大于CK,与CK相比,4种接种方式处理下的苗高、地径和总生物量增幅分别为11.48%～29.51%、3.21%～26.61%和36.30%～148.18%; 总根长、总根表面积、根平均直径、总根体积和根尖数增幅分别为70.62%～139.39%、74.70%～140.97%、164.28%～200.00%、75.00%～250.00%和28.03%～167.42%。不同接种方式处理的花榈木结瘤幼苗生理生化指标差异显著,浇灌处理的花榈木结瘤幼苗叶绿素含量、硝酸还原酶活性、硝态氮含量和根系活力都明显高于其他处理。接种上根瘤的花榈木幼苗叶片全氮、鲜瘤豆血红蛋白含量、总氮量以及固氮量明显高于CK,浇灌和浸根处理优于其他处理。【结论】花榈木根瘤接种的适宜方式为用萌发种子浸根和用菌液浇灌长出真叶后的幼苗。     

诱导杨树结瘤固氮的研究

６种植物激素（２,４－Ｄ、ＮＡＡ、ＩＡＡ、ＩＢＡ、６－ＢＡ、Ｓ．Ｌ．）用不同浓度处理杨树无性系ＮＬ－８０１０６组培苗根系,其中２,４－Ｄ、ＩＢＡ、Ｓ．Ｌ．有不同程度的诱瘤作用。以２,４－Ｄ和ＩＢＡ诱导结瘤最为稳定有效,２,４－Ｄ诱瘤的最适浓度１．０～１．５ｍｇ／Ｌ,ＩＢＡ为２．５～３．０ｍｇ／Ｌ。分离、回接和显微镜观察表明根瘤菌在诱瘤剂作用下已侵入诱导瘤。诱瘤剂在接种根瘤菌情况下对苗木生长有较为明显的促进作用,无论株高、鲜重和叶绿素含量都高于对照,尤以ＩＢＡ（３．０ｍｇ／Ｌ）最为显著。     

Sinorhizobium meliloti lsrB is involved in alfalfa root nodule development and nitrogen-fixing bacteroid differentiation

Rhizobia interact with host legumes to induce the formation of nitrogen-fixing nodules, which is very important in agriculture and ecology. The development of nitrogen-fixing nodules is stringently regulated by host plants and rhizobial symbionts. In our previous work, a new Sinorhizobium meliloti LysR regulator gene (lsrB) was identified to be essential for alfalfa nodulation. However, how this gene is involved in alfalfa nodulation was not yet understood. Here, we found that this gene was associated with prevention of premature nodule senescence and abortive bacteroid formation. Heterogeneous deficient alfalfa root nodules were induced by the in-frame deletion mutant of lsrB (lsrB1-2), which was similar to the plasmid-insertion mutant, lsrB1. Irregular senescence zones earlier appeared in these nodules where bacteroid differentiation was blocked at different stages from microscopy observations. Interestingly, oxidative bursts were observed in these nodules by DAB staining. The decreased expression of lipopolysaccharide core genes (lpsCDE) was correspondingly determined in these nodules. S. meliloti lipopolysaccharide is required for suppression of oxidative bursts or host cell defense. These findings demonstrate that the S. meliloti lsrB gene is involved in alfalfa root nodule development and bacteroid differentiation by suppressing oxidative bursts or defense responses in host cells.     

贵州省豆科结瘤固氮植物资源调查

通过对贵州省豆科植物结瘤固氮资源的调查,共采集根瘤标本242份,分属豆科植物42属103种及变种,其中蝶形花亚科、含羞草亚科、云实亚科植物的结瘤率分别为95%,75%和5%;采集的根瘤中,96%生于寄主的侧根,形状以球形、长柱形为主,颜色多为白色、褐色.对分离纯化的55株根瘤菌进行了回接试验,回接结瘤率为33%.生态条件是影响根瘤的大小、数量以及有效性的原因之一.     

Isolation and application of effective nitrogen fixation rhizobial strains on low-phosphorus acid soils in South China

Soybean (Glycine max L.) is a very important food and oil crop in China. Legume-rhizobium symbiotic nitrogen (N) fixation is an important biological character and also the base of improving soil fertility of soybean. However, soybean production and development is severely limited in tropical and subtropical areas in China due to a lack of effective rhizobial inoculants adapting to low-phosphorus (P) acid soils. In the present study, 12 soybean rhizobial strains were isolated and purified from the nodules of two soybean genotypes contrasting in P efficiency, which were grown on different low-P acid soils with different soybean cultivation histories. Results from 16S rDNA sequence analysis showed that these 12 rhizobial strains belonged to the genus of Bradyrhizobium, which had higher nitrogenase activities compared to the control strain, Bradyrhizboium japonicum USDA110. A field experiment was carried out by applying rhizobial inoculants, a mixture of three rhizobial strains that showed the highest nitrogenase activity, on a typical low-P acid soil in South China. The results showed that, without inoculation, no nodules were formed in the three soybean genotypes tested; with inoculation, the nodulation rates in all were 100%. Inoculation with rhizobial inoculants not only made many nodules formed, but also increased soybean shoot biomass and yield, and improved nitrogen (N) and P nutrient status. Among which, shoot dry weight, N and P content of a soybean genotype, Huachun 3, inoculated with rhizobium were increased 154.3%, 152.4% and 163.2% compared to that without inoculation, respectively. We concluded that: (i) The effective indigenous rhizobial strains isolated in this study from soybeans on low-P acid soils in South China have the characters of broad host range, high nodulation efficiency, efficient N fixation, great low pH and low P tolerance. (ii) Soil environment and host types are the key factors to screen the effective rhizobial strains. Considering soil pH values and P efficiency of the host genotypes might increase the screening efficiency. (iii) Improving N status and facilitating root growth might be the mechanisms of increasing the P uptake in soybean plants inoculated with the effective rhizobial strains on low-P acid soils. (iv) Inoculation with the effective rhizobial inoculants could significantly improve growth, N and P content of soybean on low-P acid soils, which might be an effective approach to enhance soybean cultivation and development in these areas. Therefore, application and extension of inoculation techniques with effective rhizobial inoculants in legumes would result in great economical, environmental and ecological benefits. The authors contributed equally to this work Supported by National Key Basic Research and Development of China (Grant No. 2005CB120902), McKnight Foundation Collaborative Crop Research Program (USA) (Grant No. 05-780) and National Natural Science Foundation of China (Grant No. 30571111)     

Shoot control of root development and nodulation is mediated by a receptor-like kinase

In legumes, root nodule organogenesis is activated in response to morphogenic lipochitin oligosaccharides that are synthesized by bacteria, commonly known as rhizobia. Successful symbiotic interaction results in the formation of highly specialized organs called root nodules, which provide a unique environment for symbiotic nitrogen fixation. In wild-type plants the number of nodules is regulated by a signalling mechanism integrating environmental and developmental cues to arrest most rhizobial infections within the susceptible zone of the root. Furthermore, a feedback mechanism controls the temporal and spatial susceptibility to infection of the root system. This mechanism is referred to as autoregulation of nodulation, as earlier nodulation events inhibit nodulation of younger root tissues. Lotus japonicus plants homozygous for a mutation in the hypernodulation aberrant root (har1) locus escape this regulation and form an excessive number of nodules. Here we report the molecular cloning and expression analysis of the HAR1 gene and the pea orthologue, Pisum sativum, SYM29. HAR1 encodes a putative serine/threonine receptor kinase, which is required for shoot-controlled regulation of root growth, nodule number, and for nitrate sensitivity of symbiotic development.     

用~(15)N 示踪法测定2,4-D 诱发的小麦和向日葵根瘤的固氮活性

用~(15)N 示踪法定性测定了经2,4-D 诱发的、接种豇豆根瘤菌(R.sp),菜豆根瘤茵(R.phaseoli)等四种根瘤菌和圆褐固氮菌(Az.chroococcum)的小麦(Triticum aesticum)和向日葵(Helianthus annuus)根瘤,结果表明具有固氮活性。     

Deregulation of a Ca2+/calmodulin-dependent kinase leads to spontaneous nodule development

Induced development of a new plant organ in response to rhizobia is the most prominent manifestation of legume root-nodule symbiosis with nitrogen-fixing bacteria. Here we show that the complex root-nodule organogenic programme can be genetically deregulated to trigger de novo nodule formation in the absence of rhizobia or exogenous rhizobial signals. In an ethylmethane sulphonate-induced snf1 (spontaneous nodule formation) mutant of Lotus japonicus, a single amino-acid replacement in a Ca2+/calmodulin-dependent protein kinase (CCaMK) is sufficient to turn fully differentiated root cortical cells into meristematic founder cells of root nodule primordia. These spontaneous nodules are genuine nodules with an ontogeny similar to that of rhizobial-induced root nodules, corroborating previous physiological studies. Using two receptor-deficient genetic backgrounds we provide evidence for a developmentally integrated spontaneous nodulation process that is independent of lipochitin-oligosaccharide signal perception and oscillations in Ca2+ second messenger levels. Our results reveal a key regulatory position of CCaMK upstream of all components required for cell-cycle activation, and a phenotypically divergent series of mutant alleles demonstrates positive and negative regulation of the process.     

2，4-D处理罗冈甜橙果实后的降解

用质量浓度为200mg/L的2，4D与45%的施宝克（杀菌剂）1000倍液组成的混合溶液浸泡罗岗甜橙果实3min，然后用薄膜袋包装果实，25摄氏度、湿度74%下贮藏，用HPLC方法分析了果实内2，4-D降解情况，结果表明:处理果实放置24H的果皮内2，4-D含量比对照果增加3.29倍；放置24h的果皮内2，4-D含量明显高于果肉。处理的果实放置24h以后，通过从果皮吸收，使果肉内2，4-D含量显著增加，贮藏5d的果肉2，4-D含量达到最高值，贮藏30d的果肉内2，4-D含量为1570.35NG/G,以后显著减少，低于农业部颁发的2，4-D残留量，符合柑橘食用安全性．