Effect of Rhizobium inoculation methodologies on nodulation and growth of Leucaena leucocephala

The aim of this study was to evaluate the effect of five methods of Rhizobium inoculum application on nodulation and nitrogen fixation in Leucaena leucocephala seedlings cultivated for 6 months in the greenhouse. Plants inoculated with alginate beads were significantly more developed and more nodulated than plants inoculated with the other methodologies used.     

Evaluation and improvement of the energy efficiency of nitrogen fixation in Lotus corniculatus nodules induced by Rhizobium loti strains indigenous to Uruguay

In order to evaluate energy efficiency of nitrogen fixation by the Lotus corniculatus/Rhizobium loti symbiosis, Uruguayan R. loti strains were tested for hydrogen-uptake (Hup) status. Nodules induced in L. corniculatus by all eight R. loti strains tested evolved high amounts of hydrogen (2.0–8.7 mol H2/h.g nodule fresh weight). This production of hydrogen corresponds to 38–69% of total nitrogenase activity estimated as acetylene reduction, suggesting that hydrogen is not recycled within these nodules. This was confirmed by the lack of hydrogenase activity in bacteroid suspensions. Additionally, no hybridization signals were observed in total DNA restriction digests from these strains when a DNA fragment containing part of hydrogenase structural genes from Rhizobium leguminosarum bv. viciae was used as probe. Cosmid pHU52, containing the complete gene cluster required for hydrogen oxidation in Bradyrhizobium japonicum, was introduced into two R. loti strains. Transconjugants from only one of the strains were able to express hydrogenase activity in vegetative cells incubated under the derepression conditions described for B. japonicum. Bacteroids induced by both transconjugant strains in L. corniculatus and Lotus tenuis expressed hydrogenase activity in nodules. The level of hydrogenase activity induced in L. tenuis nodules was two-fold higher than those induced in L. corniculatus. This implies the existence of a strong host effect on hydrogenase expression in this symbiotic system.     

The capacity of Jamaican mine spoils, agricultural and forest soils to nodulate Myrica cerifera, Leucaena leucocephala and Casuarina cunninghamiana

Soils from seven sites on the island of Jamaica were assayed for the symbiotic diazotrophs Frankia and Rhizobium using serial dilutions. Most probable number and least squares regression methods were used to estimate each soil's capacity to nodulate native Myrica cerifera, exotic Leucaena leucocephala and exotic Casuarina cunninghamiana. The sample sites included a montane forest, a slash-and-burn agricultural site, reclaimed bauxite mining areas, abandoned sugar cane fields, and a garden plot. None of the host plants used in the bioassay were present on the sites sampled except for scattered L. leucocephala on one site. Frankia capable of nodulating M. cerifera, which is native to Jamaican highlands, occurred at all sites sampled. No C. cunninghamiana-infective Frankia was detected in soils sampled. Only soils from one site on the tropical coastal plain harbored rhizobia able to nodulate L. leucocephala (37 nodulation units cm^?3 of soil). A subset of nodulated M. cerifera and L. leucocephala reduced acetylene to ethylene indicating nitrogenase activity. The slash-and-burn agricultural site, which was situated at an elevation of 200 m and possessed both high natural fertility and high soil moisture-supplying capacity, had significantly greater Myrica infectious capacity (1 000 nodulation units cm^?3 of soil) than the other sites (7?207 nodulation units cm^?3 of soil). A planned, paired comparison revealed that a recently cultivated sugar cane field and a recently reclaimed bauxite mining site together had significantly less Myrica-infective Frankia (4 nodulation units cm^?3 of soil) than a corresponding pair of sites consisting of a sugar cane field abandoned for 25 years and a bauxite mining site reclaimed 20 years before sampling (118 nodulation units cm^?3 of soil). Results indicate that Myrica-infective Frankia is widespread in Jamaica, that the number of Myrica-infective Frankia units vary from site to site in accordance with soil type and soil history, that Jamaican sites sampled lack soil Frankia populations capable of nodulating a casuarina host, that rhizobial symbionts capable of nodulating L. leucocephala may be geographically restricted to lowlands in Jamaica, and that the occurrence of Frankia in these soils is independent of host plant presence.     

Analysis of pss genes of Rhizobium leguminosarum required for exopolysaccharide synthesis and nodulation of peas: their primary structure and their interaction with psi and other nodulation genes

Summary Strains of Rhizobium leguminosarum (R. l.) biovar viciae containing pss mutations fail to make the acidic exopolysaccharides (EPS) and are unable to nodulate peas. It was found that they also failed to nodulate Vicia hirsuta, another host of this biovar. When peas were co-inoculated with pss mutant derivatives of a strain of R.l. bv viciae containing a sym plasmid plus a cured strain lacking a sym plasmid (and which is thus Nod^-, but for different reasons) but which makes the acidic EPS, normal numbers of nodules were formed, the majority of which failed to fix nitrogen (the occasional Fix⁺ nodules were pressumably induced by strains that arose as a result of genetic exchange between cells of the two inoculants in the rhizosphere). Bacteria from the Fix^- nodules contained, exclusively, the strain lacking its sym plasmid. When pss mutant strains were co-inoculated with a Nod^- strain with a mutation in the regulatory gene nodD (which is on the sym plasmid pRL1JI), normal numbers of Fix⁺ nodules were formed, all of which were occupiced solely by the nodD mutant strain. Since a mutation in nodD abolishes activation of other nod genes required for early stages of infection, these nod genes appear to be dispensable for subsequent stages in nodule development. Recombinant plasmids, containing cloned pss genes, overcame the inhibitory effects of psi, a gene which when cloned in the plasmid vector pKT230, inhibits both EPS production and nodulation ability. Determination of the sequence of the pss DNA showed that one, or perhaps two, genes are required for correcting strains that either carry pss mutations or contain multi-copy psi. The predicted polypeptide product of one of the pss genes had a hydrophobic aminoterminal region, suggesting that it may be located in the membrane. Since the psi gene product may also be associated with the bacterial membrane, the products of psi and pss may interact with each other.     

Inhibition of mycorrhizal symbiosis in Leucaena leucocephala by chlorothalonil

The effect of the fungicide, chlorothalonil, on vesicular-arbuscular mycorrhizal (VAM) symbiosis was studied in a greenhouse using Leucaena leucocephala as test plant. Chlorothalonil was applied to soil at 0, 50, 100 and 200 μg g⁻¹. The initial soil solution P levels were 0.003 μg mL⁻¹ (sub-optimal) and 0.026 μg mL⁻¹ (optimal). After 4 weeks, the sub-optimal P level was raised to 0.6 μg mL⁻¹ (high). The soil was either uninoculated or inoculated with the VAM fungus, Glomus aggregatum. The fungicide reduced mycorrhizal colonization of roots, development of mycorrhizal effectiveness, shoot P concentration and uptake and dry matter yields at all concentrations tested, although the highest inhibitory effect was noted as the concentration of the fungicide was increased from 50 to 100 μg g⁻¹. Phosphorus applied after four weeks tended to partially offset the deleterious effects of chlorothalonil in plants grown in the inoculated and uninoculated soil which suggests that the fungicide was interfering with plant P uptake. The results suggest that the use of chlorothalonil should be restricted to levels below 50 μg g⁻¹ if the benefits of mycorrhizal symbiosis are to be expected. Contribution from Hawaii Institute of Tropical Agriculture and Human Resources Journal Series No. 3464. Contribution from Hawaii Institute of Tropical Agriculture and Human Resources Journal Series No. 3464.     

银合欢叶作氮源栽培食用菌研究

以银合欢叶作氮源全部或部分取代基质中米糠或麸皮,栽培毛木耳、凤尾菇、金针菇、黑木耳、香菇和佛罗里达侧耳6种食用菌。结果表明,银合欢叶能促进菌丝生长。栽培中发现,含10%银合欢叶和17%麸皮的基质能使金针菇获得增产,而含10%或20%银合欢叶的基质可使其它5种食用菌产量提高11.4%~39.1%。     

The role of motility in the efficiency of nodulation by Rhizobium meliloti

Tn5 mutants of Rhizobium meliloti L5.30 defective in motility (Mot^-) were isolated and compared to the parent with respect to the nodulation activity. Each of the mutants was able to generate normal nodules on the alfalfa (Medicago sativa) but had slightly delayed nodule formation. Coinoculation of lucerne with wild type Mot⁺ and Mot^- cells in the wide range of ratios resulted in nodules occupied in the majority by a motile strain suggesting that motility is a factor involved in the competition for nodule formation.     

The cloned 1-aminocyclopropane-1-carboxylate (ACC) deaminase gene from Sinorhizobium sp. strain BL3 in Rhizobium sp. strain TAL1145 promotes nodulation and growth of Leucaena leucocephala

The objective of this study was to determine the role of 1-aminocyclopropane-1-carboxylate (ACC) deaminase of symbionts in nodulation and growth of Leucaena leucocephala. The acdS genes encoding ACC deaminase were cloned from Rhizobium sp. strain TAL1145 and Sinorhizobium sp. BL3 in multicopy plasmids, and transferred to TAL1145. The BL3-acdS gene greatly enhanced ACC deaminase activity in TAL1145 compared to the native acdS gene. The transconjugants of TAL1145 containing the native or BL3 acdS gene could grow in minimal media containing 1.5mM ACC, whereas BL3 could tolerate up to 3mM ACC. The TAL1145 acdS gene was inducible by mimosine and not by ACC, while the BL3 acdS gene was highly inducible by ACC and not by mimosine. The transconjugants of TAL1145 containing the native- and BL3-acdS genes formed nodules with greater number and sizes, and produced higher root mass on L. leucocephala than by TAL1145. This study shows that the introduction of multiple copies of the acdS gene increased ACC deaminase activities of TAL1145 and enhanced its symbiotic efficiency on L. leucocephala.     

含羞草素及其代谢产物的降解和银合欢去毒(综述)

本文概述含羞草素及其代谢产物降解的物理、化学和生物学途径,为银合欢的去毒研究提供依据。     

Persistence and recovery of introduced Rhizobium ten years after inoculation on Leucaena leucocephala grown on an Alfisol in southwestern Nigeria

Establishment of Leucaena leucocephala was poor at Ibadan (Transition forest-savanna zone) and Fashola (savanna zone, 70 km north of Ibadan) in southwestern Nigeria as a result of low soil fertility and the presence of only a few native rhizobia capable of nodulating it. Inoculation with L. leucocephala at these two locations in 1982 resulted in striking responses with Rhizobium strains IRc 1045 and IRc 1050 isolated from L. leucocephala grown in Nigeria. The persistence of inoculated effective Rhizobium strains after inoculation is desirable since it removes the need for reinoculation. Because of the perennial nature of L. leucocephala and its use in long-term alley farming experiments, we examined the persistence of inoculated rhizobial strains after inoculation, and their ability to sustain N₂-fixation and biomass production at Ibadan. In 1992, ten years after Rhizobium introduction, uninoculated, L. leucocephala fixed about 150 kg N ha^-1 yr^-1 or about 41% of total plant N compared to 180 kg N ha^-1 yr^-1 or 43% measured in 1982. Serological typing of the nodules using the Enzyme-Linked-Immunosorbent Assay (ELISA) and intrinsic resistance to the streptomycin test revealed that most of the nodules (96%) formed on L. leucocephala in 1992 were by Rhizobium strains IRc 1045 and IRc 1050, which were inoculated in 1982. Nodules were absent on uninoculated L. leucocephala grown on the adjacent field with no history of L. leucocephala cultivation. We conclude that the N₂ fixed by Rhizobium strains IRc 1045 and IRc 1050 persisted for many years in the absence of L. leucocephala and sustained effectively fixed N₂ which growth and yield of L. leucocephala after several years, thus encouraging a possible low-input alley farming system by smallholder farmers in Nigeria.     

Effect of Rhizobium andAzospirillum lipoferum inoculation on the nodulation,yield and nitrogen uptake of peanut cultivars

Peanut (Arachis hypogaea Linn.) Cvs. Robut 33-1 and JL 24 were inoculated with Rhizobium strain NC 92 and a strain ofAzospirillum lipoferum singly and as mixed inoculum. Seed inoculation with these bacteria enhanced nodulation, N content and yield of these cultivars under field conditions. While a mix inoculation of these two diazotrophic cultures had an adverse effect on these parameters as compare to single inoculation.     

紫云英根瘤菌胞外多糖合成基因exo1的序列分析

对互补紫云英根瘤菌Ｅｘｏ－Ｎｄｖ－Ｆｉｘ－变种的２．６ｋｂＤＮＡ片段进行序列测定。分析结果表明,该片段内存在一个完整的阅读框架（ＯＲＦ）ｅｘｏ１。ｅｘｏ１编码３４０个氨基酸,为细胞质蛋白。Ｅｘｏ１蛋白序列与苜蓿根瘤菌的糖基转移酶ＥｘｏＵ有较强的同源性。利用启动子检测质粒,分析ｅｘｏ１基因的表达,发现在ｅｘｏ１基因上有两个启动子活性区段,Ｐｅｘｏ１ａ位于基因前端,Ｐｅｘｏ１ｂ位于基因中间。Ｐｅｘｏ１ａ很可能包含了ｅｘｏ１基因的启动子。     

Propagation of the tropical tree,Leucaena leucocephala K67, by in vitro bud culture

A tissue culture method is described for clonal multiplication of Leucaena leucocephala K67 using single lateral bud explants from 2–3 m tall greenhouse grown trees. N-6 benzyladenine (BA: 3.0 mg.1^-1) and napthaleneacetic acid (NAA: 0.05 mg.1^-1) in Murashige & Skoog's (MS) medium were found to be best suited for multiple shoot differentiation in 4–5 week old cultures. Analysis of variance of the main treatment effects of BA and NAA on shoot parameters showed that BA significantly (P=0.001) affected shoot development while NAA did not. A shoot multiplication rate of 22±3.63 shoots per bud explant was obtained in 150 days on 1/2 strength MS medium with 3.0 mg.1^-1 BA and 0.05 mg.1^-1 NAA. Shoots developed adventitious roots within 15 days in 1/2 strength MS medium containing indole-3-butyric acid (IBA: 3.0 mg.1^-1) and Kinetin (0.05 mg.1^-1). Eighty percent of the transplanted plantlets are being grown in greenhouse conditions.     

应用RAPD分子标记技术研究苜蓿根瘤菌的田间竞争结瘤能力

以在温室条件筛选出与 Vector苜蓿品中匹配较好的根瘤菌系 CCBAU30 1 38和 Vector为材料 ,应用 RAPD技术研究CCBAU30 1 38田间竞争结瘤能力。结果显示 ,利用冻融法处理的根瘤、菌体提取的 DNA可以直接作为 PCR扩增的模板 ,扩增结果与以类菌体 DNA及总 DNA作为模板处理的结果相同 ;以根瘤处理物作为 PCR扩增的模板 ,应用 RAPD分子标记技术对接种菌 CCBAU30 1 38田间竞争结瘤能力进行研究 ,接种 1 4 0 d后 ,CCBAU30 1 38田间占瘤率为 4 7.7% ,表明该菌具有较强竞争结瘤能力和持久力。试验结果还说明 ,在接种菌与土著菌有差异的条件下 ,应用 RAPD技术开展竞争结瘤能力研究 ,可以直接以根瘤处理物作为 PCR扩增的模板 ,具有简易、快速、准确等优点     

Enhanced competitiveness for nodulation of Medicago sativa by Rhizobium meliloti transconjugants harbouring the root-inducing plasmids of Agrobacterium rhizogenes strain 15834

Abstract The root-inducing plasmid of Agrobacterium rhizogenes strain 15834 marked with transposon Tn5-mob with a helper plasmid RP4-4 was mobilized into nitrogen-fixing Rhizobium meliloti strain CIAM 1759. The resulting transconjugants did not induce ‘hairy’ root syndrome but developed nitrogen-fixing nodules on alfalfa. Among the 9 transconjugants tested, 6 strains had increased nodulation rates. The competitiveness of 2 of these R. meliloti (pRi) strains was significantly enhanced as compared with the parent strain CIAM 1759; this was confirmed both in tube and in pot tests.     

Enhanced competitiveness for nodulation of Medicago sativa by Rhizobium meliloti transconjugants harbouring the root-inducing plasmids of Agrobacterium rhizogenes strain 15834

Abstract The root-inducing plasmid of Agrobacterium rhizogenes strain 15834 marked with transposon Tn 5 -mob RP4-4 with a helper plasmid was mobilized into nitrogen-fixing Rhizobium meliloti strain CIAM 1759. The resulting transconjugants did not induce ‘hairy’ root syndrome but developed nitrogen-fixing nodules on alfalfa. Among the 9 transconjugants tested, 6 strains had increased modulation rates. The competitiveness of 2 of these R. meliloti (pRi) strains was significantly enhanced as compared with the parent strain CIAM 1759; this was confirmed both in tube and in pot tests.     

The role of seed reserves in arbuscular mycorrhizal formation and growth of Leucaena leucocephala (Lam.) de Wit. and Zea mays L.

We show here that seed reserves in Leucaena leucocephala (Lam.) de Wit. and Zea mays L. (maize) are important for mycorrhizal formation and seedling growth. Seed reserve removal reduced mycorrhizal formation markedly in Leucaena but not in maize, except at 15 and 45 days after seed reserve removal. Partial or total removal of seed reserves decreased plant growth and tissue nutrient concentrations in both hosts. Nodule number in Leucaena, which was related positively to plant biomass and mycorrhizal infection levels, was reduced when one or both cotyledons were severed. Leucaena seedlings without or with partial seed reserves had higher nutrient use efficiencies throughout seedling growth. But such an effect was observed only initially in maize. Seed reserve removal increased the specific absorption rates of nutrients in both hosts. Phosphorus absorption rate was significantly and positively related to root infection levels in both Leuceana and maize. Though the growth rates of plants without seed reserves were low initially, these plants had higher growth rates during later stages. We conclude that seed reserves are not only important for seedling growth, but also for mycorrhizal formation and nodulation. Received: 15 July 1999 / Accepted: 6 December 1999