Control of nodule number by the phytohormone abscisic Acid in the roots of two leguminous species

The effects of the phytohormone abscisic acid (ABA) on plant growth and root nodule formation were analyzed in Trifolium repense (white clover) and Lotus japonicus, which form indeterminate and determinate nodules, respectively. In T. repense, although the number of nodules formed after inoculation with Rhizobium leguminosarum bv. trifolii strain 4S (wild type) was slightly affected by exogenous ABA, those formed by strain H1(pC4S8), which forms ineffective nodules, were dramatically reduced 28 days after inoculation (DAI). At 14 and 21 DAI, the number of nodules formed with the wild-type strain was decreased by exogenous ABA. In L. japonicus, the number of nodules was also reduced by ABA treatment. Thus, exogenous ABA inhibits root nodule formation after inoculation with rhizobia. Observation of root hair deformation revealed that ABA blocked the step between root hair swelling and curling. When the ABA concentration in plants was decreased by using abamine, a specific inhibitor of 9-cis-epoxycarotenoid dioxygenase, the number of nodules on lateral roots of abamine-treated L. japonicus increased dramatically, indicating that lower-than-normal concentrations of endogenous ABA enhance nodule formation. We hypothesize that the ABA concentration controls the number of root nodules.     

Nodulation ofTrifolium subterraneum byRhizobium leguminosarum

Summary Four cultivars ofTrifolium subterraneum were nodulated by five strains ofRhizobium leguminosarum; all combinations except one gave 100% nodulation. Rates of nodule formation and total nodule numbers were similar to those with an effectiveR. trifolii strain. The nodules were more commonly associated with lateral roots and were ineffective in nitrogen fixation.     

First contribution to the bionomics of the pollen wasp Ceramius palaestinensis (Giordani Soika, 1957) (Hymenoptera: Vespidae: Masarinae) in Turkey

Data about the bionomics of the pollen wasp Ceramius palaestinensis (Giordani Soika, 1957) are presented for the first time. Ceramius palaestinensis is characterized by the following ethological elements: (i) the nest is excavated in non-friable soil; (ii) the burrow is surmounted by a turret from earth extracted from within the burrow; (iii) the nest is possibly perennial; (iv) the nest has a relatively short, vertical to sub-vertical main shaft with an expansion at the bottom; (v) the main shaft is not terminated by a cell; (vi) secondary shafts are absent; (vii) the cells are sub-horizontal, all at different depths; (viii) brood cells are constructed mud-cells built within an excavated cell, formed from earth excavated within the burrow; (ix) imagines visit only flowers of Trifolium (Fabaceae) and brood cells are provisioned exclusively with pollen from this plant genus; Trifolium species varying considerably in inflorescence morphology lead to different positions of foraging wasps on the inflorescences during flower visits; Trifolium resupinatum or T. clusii inflorescences with resupinate flowers function as a disc-shaped pseudanthium with the vexilla of the flowers arranged on the outer periphery serving as petal-like exhibition organs; and (x) males patrol and perch at water collection sites, at flowers and within nesting aggregations; copulations or copulatory attempts are observed there.     

Rhizobia from wild legumes: diversity, taxonomy, ecology, nitrogen fixation and biotechnology

Wild legumes (herb or tree) are widely distributed in arid regions and actively contribute to soil fertility in these environments. The N2-fixing activity and tolerance to drastic conditions may be higher in wild legumes than in crop legumes. The wild legumes in arid zones harbor diverse and promiscuous rhizobia in their root-nodules. Specificity existed only in few rhizobia from wild legumes, however, the majority of them are with wide host range. Based on phenotypic characteristics and molecular techniques (protein profiles, polysaccharides, plasmids, DNA-DNA hybridization, 16SrRNA, etc.), the root-nodule bacteria that was isolated from wild legumes had been classified into four genera (Rhizobium, Bradyrhizobium, Mesorhizobium and Sinorhizobium). The rhizobia of wild legumes in arid zones, exhibit higher tolerance to the prevailing adverse conditions, e.g. salt stress, elevated temperatures and desiccation. These rhizobia may be used to inoculate wild, as well as, crop legumes, cultivated in reclaimed desert lands. Recent reports indicated that the wild-legume rhizobia formed successful symbioses with some grain legumes. Moreover, intercropping of some N2-fixing tree legumes (e.g. Lablab, Leucaena, Sesbania, etc.) to pasture grasses improved biomass yield and herb quality. In recent years, the rhizobia of wild legumes turn the attention of biotechnologists. These bacteria may have specific traits that can be transferred to other rhizobia through genetic engineering tools or used to produce industrially important compounds. Therefore, these bacteria are very important from both economic and environmental points of view.     

Grafting between model legumes demonstrates roles for roots and shoots in determining nodule type and host/rhizobia specificity

Previous grafting experiments have demonstrated that legume shoots play a critical role in symbiotic development of nitrogen-fixing root nodules by regulating nodule number. Here, reciprocal grafting experiments between the model legumes Lotus japonicus and Medicago truncatula were carried out to investigate the role of the shoot in the host-specificity of legume-rhizobia symbiosis and nodule type. Lotus japonicus is nodulated by Mesorhizobium loti and makes determinate nodules, whereas M. truncatula is nodulated by Sinorhizobium meliloti and makes indeterminate nodules. When inoculated with M. loti, L. japonicus roots grafted on M. truncatula shoots produced determinate nodules identical in appearance to those produced on L. japonicus self-grafted roots. Moreover, the hypernodulation phenotype of L. japonicus har1-1 roots grafted on wild-type M. truncatula shoots was restored to wild type when nodulated with M. loti. Thus, L. japonicus shoots appeared to be interchangeable with M. truncatula shoots in the L. japonicus root/M. loti symbiosis. However, M. truncatula roots grafted on L. japonicus shoots failed to induce nodules after inoculation with S. meliloti or a mixture of S. meliloti and M. loti. Instead, only early responses to S. meliloti such as root hair tip swelling and deformation, plus induction of the early nodulation reporter gene MtENOD11:GUS were observed. The results indicate that the L. japonicus shoot does not support normal symbiosis between the M. truncatula root and its microsymbiont S. meliloti, suggesting that an unidentified shoot-derived factor may be required for symbiotic progression in indeterminate nodules.     

Rhizobium trifolii mutants deficient in exopolysaccharide production

Spontaneous mutants of Rhizobium trifolii 24AR5 which did not produce exopoly-saccharide were isolated. The non-mucoid mutants formed small white and ineffective nodules on both red and white clover. These nodules contained infection threads, but only a small number of bacteria were released into nodule cells, and bacteroids were rarely observed. The non-mucoid phenotype was not complemented by the symbiotic plasmid (pJB5JI) of Rhizobium leguminosarum.     

Factors contributing to the loss of seed from the seed-bank of Trifolium balansae and Trifolium resupinatum over summer

Abstract The influence of predation, fungi and germination of seed of the annual legumes Trifolium balansae and Trifolium resupinatum was investigated by placing known quantities of seed in bags in the field; combinations of all three factors were imposed. It was found that rainfall was the most important factor contributing to the loss of seed over summer (up to 76%), followed by predation (up to 15%); fungi had only a very small influence (0.1–0.2%). Predation contributed to a larger percentage of loss over summer in T. resupinatum line SA14433 than in T. balansae cv. Paradana, which is attributed to seed size. The effect of summer grazing on the size of the seed-bank was investigated. It was found that summer grazing did not influence the amount of seed present in the seed-bank in March. Removal of all surface material in December resulted in a significant drop in seed reserves compared with the ungrazed or grazed areas. Hardseededness in March was not affected by any of the treatments; the seedling density after regeneration was not affected by summer grazing, but was reduced when all surface material was removed. The data were incorporated in a model to describe seed loss from the seed bank over summer quantitatively; the utility of such models is discussed.     

Competition studies withRhizobium trifolii in laboratory experiments

Summary Competition studies were carried out in soil cores by comparing the nodule forming ability of antibiotic resistance marked strains, inoculated at three inoculum levels onTrifolium repens versus an effective indigenous Rhizobium population of 1.5×10⁵/gm. It was seen that the indigenous marked isolate G1067 formed a high proportion of nodules sampled (>90%) at all three inoculum levels (10⁵, 10⁷ and 10⁹ cells/seed) wherein the introduced foreign strain G1032 formed >50% of the nodules at the highest inoculum level.In a test tube experiment, competition for nodule sites was examined by inoculating mixtures of twoR. trifolii strains at different input levels on two cultivars ofTrifolium repens var. Huia and Titan. It was seen that G1032 was less competitive than G1006 and G1067 on cv. Huia but was more competitive on cv. Titan than the other two strains.     

The comparative phosphorus requirements of some temperate perennial legumes

The phosphorus (P) responses of seven temperate perennial pasture legumes and two species of lupins were compared in a field trial over a range of nine P rates, from 0 to 800 kg ha^-1. The two lupins produced more than 5 t ha^-1 of dry matter in the absence of added P and showed no response to the fertiliser. In contrast, the pasture legumes initially failed to grow without added P and responded to applications of between 200 and 800 kg ha^-1. At the higher rates of P, dry matter production of the pasture legumes was equivalent to that of the lupins.In the first 2 years of the trial; the most productive pasture legume species at the higher rates of added P were also the most productive at the lower rates. Phosphorus requirements for 90% of maximum yield varied greatly between species, but were closely related to maximum yield. Thus species with low P requirements for maximum yield were not necessarily P-efficient species. In the third and subsequent years of the trial Lotus corniculatus performed better than the other pasture legumes at the lower rates of added P. In contrast to other studies Lotus pedunculatus showed no ability to outyield Trifolium repens at low rates of P. Critical P concentrations of the pasture species for the late spring-early summer period declined in the order Trifolium repens (0.34%) > Lotus pedunculatus (0.30%) > Triofolium pratense (0.28%) > Trifolium hybridum (0.27%) > Trifolium ambiguum (0.26%) > Lotus corniculatus (0.23%).Mineralisable nitrogen (N) levels were determined in soils under three species in the 7th year of the trial. At the lowest rates of added P, mineralisable N levels were much higher under Lupinus polyphyllus than under Trifolium repens or Lotus corniculatus. With increasing P rate, levels under the latter species increased, and at 100 kg P ha^-1 were equivalent to those under the lupin with no added P.     

Symbiosis between the nodule bacterium Sinorhizobium meliloti and alfalfa (Medicago sativa) under salinization conditions

Two hundred forty-three isolates of alfalfa nodule bacteria (Sinorhizobium meliloti) were obtained from legume nodules and soils sampled in the northern Aral region, experiencing secondary salinization. Isolates obtained from nodules (N isolates) were significantly more salt-tolerant than those from soils (S isolates) when grown in a liquid medium with 3.5% NaCl. It was found that wild species of alfalfa, melilot, and trigonella preferably formed symbioses with salt-tolerant nodule bacteria in both salinized and nonsalinized soils. Only two alfalfa species, Medicago falcata and M. trautvetteri, formed efficient symbioses in soils contrasting in salinity. The formation of efficient symbiosis with alfalfa in the presence of 0.6% NaCl was studied in 36 isolates (N and S) differing in salt tolerance and symbiotic efficiency. Fifteen isolates formed efficient symbioses in the presence of salt. The increase in the dry weight of the plants was 25-68% higher than in the control group. The efficiency of symbiotic interaction under salinization conditions depended on the efficiency of the isolates under standard conditions but did not correlate with the source of nodule bacteria (soil or nodule) or their salt tolerance. The results indicate that nodule bacterium strains forming efficient symbioses under salinization conditions can be found.     

花生根瘤菌Bradyrhizobium sp.MM6 Ⅲ型分泌系统的结构和功能

【目的】探究花生根瘤菌Bradyrhizobium sp.MM6的Ⅲ型分泌系统(T3SS)的结构及其在根瘤菌与不同宿主建立共生关系中的作用。【方法】同源比对分析菌株MM6的T3SS基因簇的结构特征,并采用三亲本接合转移的方法构建T3SS调节基因ttsI突变菌株;通过蛭石结瘤和石蜡切片实验,比较突变体与野生型的共生固氮表型差异。【结果】经预测,MM6的T3SS基因簇编码区长约34.1 kb,可分为3个区域,包含10个保守结构基因和8个效应蛋白基因,与B.diazoefficiens USDA110相应基因的序列相似性为83%–93%;成功构建了MM6的ttsI突变株;ttsI突变株与野生型分别与花生(S523和Y45)、野大豆和大豆中黄57结瘤,ttsI突变体在花生中的总瘤数显著增加(P<0.05),根瘤中含菌细胞更多;ttsI突变体在野大豆中平均每株植物增加4个根瘤,根瘤中含菌细胞更多,地上部干重相比野生型MM6显著增加(P<0.05);在大豆中黄57中,野生型MM6能形成红色的有效根瘤,ttsI突变体不结瘤,且植株叶片发黄,地上部干重相比野生型MM6显著降低(P<0.05)。【结论】MM6的T3SS在花生和野大豆共生体系中起着有害的作用,而在大豆中黄57的共生体系中起着有利的作用。     

Structure of nitrogen-fixing nodules formed by Rhizobium on roots of Parasponia andersonii Planch.

The structure of nitrogen-fixing nodules produced by Rhizobium infection of the non-legume Parasponia andersonii was examined by light and electron (both SEM and TEM) microscopy. Comparisons were made with the nodules previously described on P. rugosa. Like the nodules on different non-legumes formed by other types of endophytes, the Rhizobium nodules on Parasponia resembled modified roots by having a central vascular bundle surrounded by an endophyte-infected zone. The intimate association between the Rhizobium and the host nodule cell was compared with the Rhizobium association found in legumes. The rhizobia were not released from the infection thread as happens in the legume. The infection thread, which propagates the Rhizobium infection to new cells, was transformed within a nodule cell from a darkly stained (light microscopy) or very electron-dense (TEM) structure to a number of thread types. The walls of the threads varied greatly in thickness and often the thread structures were without rigid walls and were only enclosed by a plasma membrane. If the rhizobia are transformed into bacteroids, as in the legumes, it would have to occur when the threads had reached their mature size, when bacterial division had ceased. Nitrogen fixation was considered to occur in all thread types.     

Genetic consequences of a single-founder population bottleneck in Trifolium amoenum (Fabaceae)

We investigated the genetic consequences of a single-founder bottleneck in a population of showy Indian clover (Trifolium amoenum), a species presumed to be extinct until rediscovered near Occidental, California, in 1993. Electrophoretic variation was evaluated in the bottlenecked population and in a larger population (Dillon Beach) discovered during the course of this study, as well as in populations of two closely related species, T. albopurpureum var. dichotomum and T. macraei. We found a surprisingly high amount of polymorphism in the single-founder T. amoenum population from Occidental (15% of loci polymorphic; an average of 1.1 alleles per locus). However, this represents a 53% reduction in number of polymorphic loci and a 20% reduction in average number of alleles per locus compared to three Trifolium populations with putatively similar mating systems (the Dillon Beach T. amoenum population and both populations of T. albopurpureum var. dichotomum). Expanding the genetic base of the Occidental T. amoenum population is a priority due to concerns about loss of evolutionary potential and the possibility of deleterious effects associated with inbreeding. However, using seed from the Dillon Beach T. amoenum population may not be beneficial due to distinct, presumably adaptive differences between plants from the two populations and concerns about outbreeding depression.     

Newly featured infection events in a supernodulating soybean mutant SS2-2 by Bradyrhizobium japonicum

Supernodulating soybean (Glycine max L. Merr.) mutant SS2-2 and its wild-type counterpart, Sinpaldalkong 2, were examined for the microstructural events associated with nodule formation and development. SS2-2 produced a substantially higher percentage of curled root hairs than the wild type, especially at 14 days after inoculation with Bradyrhizobium japonicum. In addition, there was new evidence that in SS2-2, B. japonicum also entered through fissures created by the emerging adventitious root primordia. Early steps of nodule ontogeny were faster in SS2-2, and continued development of initiated nodules was more frequent and occurred at a higher frequency than in the wild type. These data suggest that the early expression of autoregulation is facilitated by decreasing the speed of cortical cell development, leading to the subsequent termination of less-developed nodules. The nodules of SS2-2 developed into spherical nodules like those formed on the wild type. In both the wild type and supernodulating mutant, vascular bundles bifurcate from root stele and branch off in the nodule cortex to surround the central infected zone. These findings indicate that SS2-2 has complete endosymbiosis and forms completely developed nodule vascular bundles like the wild type, but that the speed of nodule ontogeny differs between the wild type and SS2-2. Thus, SS2-2 has a novel symbiotic phenotype with regard to nodule organogenesis.     

Nodule formation and haemoglobin content in some species of Papilionaceae

Summary Characteristics of nodule formation in eleven genera of the Leguminosae, belonging to the tribes Galegeae, Genisteae, Hedysareae and Phaseoleae of the sub-family Papilionaceae are described. Variation existed in the type, size and weight of nodules formed on the legumes when inoculated with effective cowpea rhizobia, in field conditions of plant growth. Among the legumes, the haemoglobin content of nodules indicated their possible effectiveness. Dolichos lablab L., had a higher haemoglobin content per unit nodule volume than other legumes. This host may have a greater potential than the other species in symbiotic activity with legume bacteria.     

Coexistence of Burkholderia, Cupriavidus, and Rhizobium sp. nodule bacteria on two Mimosa spp. in Costa Rica

rRNA gene sequencing and PCR assays indicated that 215 isolates of root nodule bacteria from two Mimosa species at three sites in Costa Rica belonged to the genera Burkholderia, Cupriavidus, and Rhizobium. This is the first report of Cupriavidus sp. nodule symbionts for Mimosa populations within their native geographic range in the neotropics. Burkholderia spp. predominated among samples from Mimosa pigra (86% of isolates), while there was a more even distribution of Cupriavidus, Burkholderia, and Rhizobium spp. on Mimosa pudica (38, 37, and 25% of isolates, respectively). All Cupriavidus and Burkholderia genotypes tested formed root nodules and fixed nitrogen on both M. pigra and M. pudica, and sequencing of rRNA genes in strains reisolated from nodules verified identity with inoculant strains. Inoculation tests further indicated that both Cupriavidus and Burkholderia spp. resulted in significantly higher plant growth and nodule nitrogenase activity (as measured by acetylene reduction assays) relative to plant performance with strains of Rhizobium. Given the prevalence of Burkholderia and Cupriavidus spp. on these Mimosa legumes and the widespread distribution of these plants both within and outside the neotropics, it is likely that both beta-proteobacterial genera are more ubiquitous as root nodule symbionts than previously believed.     

The diversity of Rhizobia, Sinorhizobia and novel non-Rhizobial Paenibacillus nodulating wild herbaceous legumes

The objective of the present study was to isolate and characterize nodulating bacteria associated with wild legumes. For this purpose, we recovered twenty isolates from root nodules of five wild legume species: Melilotus alles, Melilotus officinalis, Trifolium pratense, Trifolium repens and Medicago sp. Most of the isolates were morphologically analogous with only few exceptions in colony shape, appearance and incubation time. All isolates were Gram negative except T.P2-4. Random amplification of polymorphic DNA showed genetic variation among isolates. The 16S rRNA sequence analysis revealed these isolates as Rhizobium, Sinorhizobium and Paenibacillus. Each of these was also screened for nod D and nod F genes with marked variation at these loci; however, the nucleotide sequence analysis confirmed the presence of nod genes. The assignment of strains to their hosts revealed a unique symbiotic association of Paenibacillus sp. nodulating T .pratense which is being reported here for the first time.     

几种生态因素对西北干旱地区豆科植物结瘤固氮的影响

通过对西北干旱地区栽培和野生豆科植物不同环境条件固氮状况的调查表明,栽培豆科植物一般能自然结瘤,野生豆科植物种的结瘤率极低。根瘤颜色栽培植物多为粉红色,而野生植物多为白色、黄色或棕色。通过对水分、光照强度和温度等不同条件下根瘤ARA测定,表明根瘤固氮活性与生态条件关系密切,而土壤水分是限制根瘤固氮活性表达的主要因素。