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1.
Rhizobia pass through the host root surroundings before they infect the root tissue. Therefore, the environmental condition of rhizosphere is one of the greatest factors in the life of Rhizobia in the soil. It was observed that the nodule number produced on the root of the legume is not proportional to the density of Rhizobia inoculated, but nearly constant within some range. The experience presented in the previous paper showed that one or ten cells of Rhizobia inoculated in a test tube were sufficient to produce a number of nodules on the host plants grown in the tube. Such facts strongly suggest the multiplication of Rhizobia in the soil before infection into tissue.  相似文献   

2.
Promiscuous soybean lines have been bred on the basis that they would nodulate freely without artificial inoculation. However, our recent studies have demonstrated that the indigenous rhizobia are not able to meet their full nitrogen (N) requirement. Rhizobia inoculation might be necessary. We examined the competition for nodule formation among native Rhizobia spp. and two inoculated Bradyrhizobia strains (R25B indigenous strain and a mixture of R25B+IRj 2180A indigenous strain from soybean lines in the savanna of northern Nigeria), their effect on N fixation, and their contribution to the yield of four soybean cultivars, grown in the field in three different agroecological zones in the moist savanna of Nigeria. About 34% of nodules were formed by the mixture of introduced R25B+IRj 2180A, while R25B formed only about 24% of the nodules but did not influence biomass and grain yield production. The indigenous rhizobia strains that nodulated the soybean varieties fixed up to 70% of their accumulated total N, confirming the promiscuous nature of these soybean varieties. Even though these varieties fixed about 75 kg N ha -1; this was not enough to sustain their optimum grain yield, as earlier reported. However, the grain yield from inoculated soybean was not significantly higher than that from the uninoculated soybean, showing a degree of competitiveness among the introduced rhizobial strains and the native rhizobia population.  相似文献   

3.
The present study was conducted to isolate and characterize rhizobial strains from root nodules of cultivated legumes, i.e. chickpea, mungbean, pea and siratro. Preliminary characterization of these isolates was done on the basis of plant infectivity test, acetylene reduction assay, C-source utilization, phosphate solubilization, phytohormones and polysaccharide production. The plant infectivity test and acetylene reduction assay showed effective root nodule formation by all the isolates on their respective hosts, except for chickpea isolate Ca-18 that failed to infect its original host. All strains showed homology to a typical Rhizobium strain on the basis of growth pattern, C-source utilization and polysaccharide production. The strain Ca-18 was characterized by its phosphate solubilization and indole acetic acid (IAA) production. The genetic relationship of the six rhizobial strains was carried out by random amplified polymorphic DNA (RAPD) including a reference strain of Bradyrhizobium japonicum TAL-102. Analysis conducted with 60 primers discriminated between the strains of Rhizobium and Bradyrhizobium in two different clusters. One of the primers, OPB-5, yielded a unique RAPD pattern for the six strains and well discriminated the non-nodulating chickpea isolate Ca-18 from all the other nodulating rhizobial strains. Isolate Ca-18 showed the least homology of 15% and 18% with Rhizobium and Bradyrhizobium, respectively, and was probably not a (Brady)rhizobium strain. Partial 16S rRNA gene sequence analysis for MN-S, TAL-102 and Ca-18 strains showed 97% homology between MN-S and TAL-102 strains, supporting the view that they were strains of B. japonicum species. The non-infective isolate Ca-18 was 67% different from the other two strains and probably was an Agrobacterium strain.  相似文献   

4.
Chickpea (Cicer arietinum L.) nodulation variants of two cultivars ICC 4948 and ICC 5003 were used as trap plants to isolate 385 native rhizobia from CCS Haryana Agricultural University, Hisar farm soil. After authentication and considering growth characteristics, selected 110 rhizobia revealed immense molecular diversity using the profiles of DNA fragments generated by Polymerase chain reaction (PCR) with enterobacterial repetitive intergeneric consensus (ERIC) sequences. Low nodulating variants of cvs ICC 4948 and ICC 5003 were able to trap more numbers of rhizobial genotypes, namely seven as compared four to five by high nodulating variants of these cultivars. Overall eight rhizobial genotypes were trapped by the chickpea cultivars. Rhizobial isolates from same nodule or same plants were present in the same or different clusters and few isolates showed 100% similarity also. Based on nodules from a plant, nodulation variant or cultivar, rhizobia could not be differentiated and no exclusive cluster was formed by either rhizobial isolates from low or high nodulating variants of both the cultivars. Two most efficient rhizobial isolates LN 707b and LN 7007 were characterized by amplification and sequencing of 16S rRNA gene. Rhizobial isolate LN 707b showed more than 98% similarity with Mesorhizobium sp SH 2851 and Mesorhizobium mediterraneum. Another isolate LN 7007 showed more than 99% similarity with the sequence of 16S r RNA gene of Mesorhizobium sp STM 398, and M. mediterraneum. So the chickpea rhizobia from Northern Indian subcontinent are proposed to be kept under M. mediterraneum strain LN707b and LN 7007.  相似文献   

5.
陈传君  金鹭  林华  胡滨  韩国全  陈世界  张婧  安微 《核农学报》2020,34(12):2762-2768
为了研究干制加工羊肉基因组DNA的最佳提取方法,本试验采用传统酚-氯仿法、磁珠法、改良CTAB法、离心柱法分别提取干制处理后的羊肉基因组DNA,并对4种方法提取的羊肉基因组DNA浓度、纯度、完整性以及提取所需时间、PCR扩增效果等进行比较。结果表明,采用磁珠法提取DNA的效果更好,DNA浓度为118.87 ng·μL-1,A260/A280值为1.89,而且此方法具有提取时间短、效率高、污染小等特点。本研究结果为干制加工羊肉基因组DNA的大批量提取和检测提供了参考依据。  相似文献   

6.
This study tested the competitive ability of three locally isolated Cyclopia rhizobia and strain PPRICI3, the strain currently recommended for the cultivation of Cyclopia, a tea-producing legume. Under sterile glasshouse conditions, the three locally isolated strains were equally competitive with strain PPRICI3. In field soils, the inoculant strains were largely outcompeted by native rhizobia present in the soil, although nodule occupancy was higher in nodules growing close to the root crown (the original inoculation area). In glasshouse experiments using field soil, the test strains again performed poorly, gaining less than 6% nodule occupancy in the one soil type. The presence of Cyclopia-compatible rhizobia in field soils, together with the poor competitive ability of inoculant strains, resulted in inoculation having no effect on Cyclopia yield, nodule number or nodule mass. The native rhizobial population did not only effectively nodulate uninoculated control plants, they also out-competed introduced strains for nodule occupancy in inoculated plants. Nonetheless, the Cyclopia produced high crop yields, possibly due to an adequate supply of soil N.  相似文献   

7.
The symbiosis between the soil bacteria Rhizobium, Sinorhizobium, Azorhizobium, Mesorhizobium or Bradyrhizobium and leguminous plants is characterised by a specific multistep signal exchange. Only when a compatible rhizobial strain encounters its leguminous host, nodules will be formed on the roots of the host. During infection of this nodule, the microsymbiont evolves into a bacteroid form which, when provided with plant-derived carbon sources, is able to convert atmospheric nitrogen to ammonia that subsequently is supplied to the plant. The developmental programme underlying nodule organogenesis and functioning has been studied intensively for several decades. In this review, several observed plant phenotypes resulting from an ineffective symbiosis between plants and mutant rhizobial strains are represented. Besides the influence of the bacterial nodulation, nitrogen fixation and surface polysaccharide genes on symbiosis, the role of other genes important for the formation of effective nitrogen fixing nodules will be explained.  相似文献   

8.
The inability to utilize a fungal siderophore as source of iron nutrition by most of the rhizobial cultures isolated from pigeon pea, could be considered a negative fitness factor since hydroxamate siderophores are found in significant amounts in natural soils. Thus these cultures were engineered to use ferrichrome a prototype of hydroxamate type siderophore. Pigeon pea Rhizobium spp. IC3123 and ST1 harboring Escherichia coli fhuA gene, responsible for uptake of Fe3+-ferrichrome, were obtained by transformation with pGR1, a broad host range plasmid carrying the fhuA gene under the control of the lac promoter of E. coli. Expression of fhuA in transformed rhizobial strains IC3123::pGR1 and ST1::pGR1 was confirmed by the ability of the plasmid-bearing strains to utilize iron bound to ferrichrome. Inoculation of pigeon pea plants with fhuA expressing rhizobial strains in pot experiments showed a significant increase in plant growth as well as nodule density as compared to those inoculated with the parent as well as the empty vector-bearing strain. Inoculation of pigeon pea seedlings with IC3123::pGR1 and ST1::pGR1 led to marked increase in shoot fresh weight, nodule number per plant, chlorophyll content of leaves and effective nodule symbiosis when compared with plants inoculated with the parent strains IC3123 and ST1. The positive effect of IC3123::pGR1 and ST1::pGR1 treatment on plant growth was more significantly observed when ferrichrome producing Ustilago maydis, known to secrete ferrichrome, was co-inoculated along with the transformed rhizobia. The presence of fhuA gene in rhizobial strains also led to an increased survival and root colonization.  相似文献   

9.
The improvement of common bean production requires the selection of effective rhizobia strains and Phaseolus vulgaris genotypes adapted to available soil phosphorus limitations. The interactions between bean genotypes and rhizobia were studied in hydroponic culture using six genotypes and four strains, CIAT899 as reference and three strains isolated from nodule of farmer's fields in the Marrakech region. The phosphorus (P) sub-deficiency caused a significant reduction on shoot biomass in some bean genotype-rhizobia combinations. Nodule biomass is significantly more reduced under P limitation for several combinations tested. Bean plants inoculated with these local rhizobial strains showed higher nodulation and an increase of nodules phytase and phosphatase activities under phosphorus sub-deficiency especially for RhM11 strain. It was concluded that the studied bean-rhizobia symbiosis differ in their adaptation to phosphorus sub-deficiency and the nodule phosphatases and phytases activities may constitute a strategy of nodulated bean plants to adapt their nitrogen fixation to P deficiency.  相似文献   

10.
The concentration of free DNA in soils has to be determined in order to understand the fate and the transport of extracellular DNA. A protocol for the extraction and determination of free DNA was developed. The procedure uses separation steps, i.e. centrifugation and ultra‐filtration. The free dsDNA was stained with PicoGreen® and determined fluorimetrically. Samples from different soils, different soil horizons, soil waters and under different land use systems were analyzed. It was found that in nearly all samples free DNA was detectable. Free DNA concentrations of up to 1950 ng (g dried sample)—1 could be detected depending on depth, soil type and system of land use.  相似文献   

11.
The aim of this work was to evaluate the competitive ability between Rhizobium leguminosarum bv trifolii strain U204 used as commercial inoculants in Uruguay for Trifolium repens L. and Trifolium pratense L. and two native strains isolated from inoculated pastures of T. pratense. T126 is an efficient nitrogen fixer and a melanin producer strain; T70 is inefficient and a melanin non-producer strain; and U204 is very efficient in both hosts but is a melanin non-producer strain. Competitiveness between the strains was determined in experiments in pots and in growth pouches under controlled conditions. In the last experiment, we evaluated pH of plant nutrient solution and inoculum ratios. Plant dry weight was determined, and the identification of nodule bacteria was done using melanin production and DNA fingerprinting (GTG5-PCR). The U204 symbiotic efficiency was not affected by the co-inoculation with the others two native strains. The T70 strain was a poor competitor when was co-inoculated with one of the effective strains in both experiments. Our results confirmed a “selective nodulation” because an effective symbiosis occurred preferentially over an ineffective one in Trifolium species. The native effective strain competed with U204 for nodule formation in both clovers species, but the nodule occupancy depended on the inoculum ratio. The pH of nutritive solution did not affect competition ability of the studied strains. It may be possible to isolate efficient, competitive, and genetically different native rhizobial strains to be used as inoculant strains for clover pastures in Uruguay. Both (GTG)5-PCR and melanin production were useful methods to identify nodulating bacteria in competition studies.  相似文献   

12.
Summary Axenically grown alfalfa (Medicago sativa L. var. Peace) was simultaneously inoculated with Canadian commercial Rhizobium meliloti strains NRG-185 and BALSAC. The plants were grown for 7 weeks in sealed units at five different root temperatures (8°, 13°, 17°, 21°, and 25°C) and at a relatively constant air temperature (24°–30°C). Nodule occupancy by each strain was determined by enzyme-linked immunosorbent assay (ELISA). Nitrogenase activity, nodule fresh weight, and plant dry weight were also measured. The lowest root-temperature regime (8°C) resulted in substantially lower nodule numbers and weights, and plant dry weights, than the higher temperature regimes. Development of nitrogenase activity was completely inhibited at 8°C. The immunoassay of nodule-strain occupancy showed markedly different strain-nodulation responses to the various root-temperature regimes. At 8°C, 63% of nodules were occupied by both strains. Dual strain occupancy decreased from 63% to 2% with increasing root-growth temperature, while the proportion of nodules containing only strain NRG-185 increased from 9% to 75%. Nodules containing only strain BALSAC remained relatively constant at 25% from 8° to 21°C, decreasing slightly at 25°C. The results suggest that root-environment temperatures during the period of nodule formation may have major differential effects on the success of competing rhizobial strains. If this is so, then selection of Rhizobium strains with enhanced low-temperature nodulation capabilities should be possible.  相似文献   

13.
Since Mycobacterium chlorophenolicum strain PCP-1 is not detectable in soil by selective plating, a specific tracking method was based on the polymerase chain reaction (PCR) using soil DNA as a target. A direct extraction protocol based on bead beating was adapted and used to obtain PCR-amplifiable DNA from five different soils. In one soil, the disruption of cells of PCP-1, of Pseudomonas fluorescens R2f and of Paenibacillus azotofixans P3L5, as well as of the indigenous bacteria increased with increasing bead beating times. After 4.5 min, lysis efficiency was about 90% or more in all cases. Total DNA yields varied between soils, from 2 to 35 μg g–1. The purification steps needed to obtain amplifiable DNA were different per soil. To detect target DNA specifically in bacterial cells, a new indirect extraction protocol was developed, which efficiently dislodged bacterial cells from the soil matrix, and produced amplifiable DNA with high yield. To detect strain PCP-1 in soil, 16S ribosomal gene-based PCR combined with oligonucleotide hybridization was applied using a most-probable-number (MPN) set-up, whereas immunofluorescence was used for calibration. Strain PCP-1 was detected shortly after introduction into three soils at about the inoculum levels, as evidenced by both approaches. Both the direct and indirect DNA extraction methods yielded similar MPN estimates. The dynamics of M. chlorophenolicum PCP-1 was estimated in two soils over 14 days via MPN-PCR/oligonucleotide probing. PCP-1 showed good survival in both soils, and results obtained by MPN-PCR with directly and indirectly extracted DNA were internally consistent. Immunofluorescence cell enumerations supported the gross stability of PCP-1 in these two as well as in two additional soils. Received: 8 February 1996  相似文献   

14.
Abstract

Nodulation and subsequent nitrogen fixation are important factors that determine the productivity of soybean (Glycine max L.). The beneficial effects of nodulation can be enhanced when rhizobial inoculation is combined with plant-growth-promoting bacteria (PGPB). The PGPB strain Bacillus thuringiensis-KR1, originally isolated from the nodules of Kudzu vine (Pueraria thunbergiana), was found to promote growth of soybean plants (variety VL Soya 2) under Jensen's tube and growth pouch conditions, when co-inoculated with Bradyrhizobium japonicum-SB1. Co-inoculation with Bacillus thuringiensis-KR1 (at a cell density of 10 cfu) provided the highest and most consistent increase in nodule number, shoot weight, root weight, root volume, and total biomass, over rhizobial inoculation and control, under both conditions. The results demonstrate the potential benefits of using nonrhizobial nodule occupants of wild legumes for the co-inoculation of soybean, with Bradyrhizobium japonicum-SB1, in order to achieve plant-growth promotion and increased nodulation.  相似文献   

15.
Background, aim and scope  An improving knowledge of bacterial community within natural environments including forest soils and leaf litters requires extraction of nucleic acids directly from environmental samples since molecular approaches provide less biased access to a larger portion of uncultivable microorganisms. However, when DNA was extracted successfully from these samples, it might still have been difficult to apply it as a template for polymerase chain reaction (PCR) amplifications due to the effect of PCR inhibitors. Various compounds from plant tissues including polysaccharides, phenolic compounds and especially humic acids can inhibit PCR amplification. Some of these inhibitors could inhibit PCR amplification by chelating the Mg2+ (cofactor for Taq polymerase), or by binding to target DNA, and PCR amplification would consequently be interfered with. Therefore, eliminating the effects of these PCR inhibitors is one of the most important steps for PCR-based molecular techniques. Four different methods were assessed in this study to purify the genomic DNA extracted from F, L layer leaf litters and forest soil in an exotic pine plantation of southeast Queensland, Australia. Materials and methods  Three samples including two leaf litters and one forest soil were collected with a core (25 × 40 cm) from a 22-year-old slash pine plantation in southeast Queensland, Australia. The DNA fragments were extracted directly using the Ultra Clean™ Mega Prep Soil DNA kit (Mo Bio Labs, Solana Beach, CA). Then, four different purification methods were applied and compared to purify the DNA for PCR amplification, which include PVPP, Sephadex TM spin column, low-melting agarose gel and a new modified gel purification method. The purified DNA from these four purification methods was detected by agarose gel electrophoresis, and the purity and usefulness of DNA samples were ultimately determined by successful PCR amplifications. Results and discussion  The DNA was extracted from each sample using the Ultra Clean™ Mega Prep Soil DNA kit, and the DNA eluents were dark in colour and sometimes formed compact aggregates. Subsequently, PCR amplification from such samples failed, although a series of dilutions had been made from neat to 1:103. The DNA purification step could not, therefore, be avoided. It was observed that both the colour of eluent and the DNA concentration decreased gradually after elution. Considering the difficulties of removing PCR inhibitors and the possibility of high DNA losses, 50–200 μl of sample DNA was used for purification. Four DNA purification methods (the PVPP spin column, Sephadex™ spin column, low-melting agarose gel and the modified gel purification method) were applied and compared on leaf litter and soil samples. The DNA purified by the modified gel purification method provided the best PCR products for 16S rRNA gene amplification, but the other methods, PVPP, Sephadex™ spin column and low-melting agarose gel, produced very weak or no products. Thus, in this study, DNA fragments which were purified by the modified gel purification method were amplified efficiently. This may be attributed to running the low-melting agrose gel for a longer time, which could remove substantial humic substances and also some other compounds from the samples and, thus, prevent them from being involved in PCR amplification. Conclusions  A new modified gel purification method which can improve DNA purification and PCR amplification of environmental DNA is first introduced in this study. Comparing PVPP, Sephadex ™ spin column, low-melting agarose gel and modified gel purification method for the effect of DNA purification, the modified gel purification method is more successful in removing the PCR amplification inhibitors and obtaining the highly purified PCR amplifiable high-molecular-weight DNA. The method described here is cheap, fast and easy to operate. It suggests in this study that the method containing less and easier following steps should be widely used to relieve the heavy working load of molecular-biological researchers. Recommendations and perspectives  This study introduces a new modified DNA purification method, and it is found that this modified gel purification method is effective in removing the PCR inhibitors and obtains highly purified DNA from leaf litters for PCR amplification. The modified gel purification method may have wider applications, although it was only assessed on leaf litter and soil samples. The effect of the modified gel purification method on the DNA purification would need to be further investigated on a variety of samples which suffered from PCR inhibitors, such as clinical samples, plant tissues and environmental samples.  相似文献   

16.
It is well known that nitrogen fixation is mainly dependent upon the combination between rhizobial strain and host plant species, though it is also affected by the environmental condition. The site of association between the host and rhizobia is the root nodule. It is, therefore, quite natural that special attention has hitherto been focused on it. A typical symbiotic relationship is going on in effective nodule, while a parasitic relationship prevails in ineffective one. Between such types of nodules there must be some differences with respect to the inner structure as well as the metabolism in them. Experiments were made to compare the morphological change of rhizobia, the amount and distribution of starch, the kinds of free amino acids in effective nodule with those in ineffective one. To obtain materials, soybean, pea, red clover, and genge (Astragalus sinicus L.) Were used with their own effective and ineffective strains of rhizobia, respectively.  相似文献   

17.
Seventy-six rhizobial isolates belonging to four different genera were obtained from the root nodules of several legumes (Vicia sativa, Vicia faba, Medicago sativa, Melilotus sp., Glycine max and Lotus corniculatus). The action of five commonly used herbicides [2,4-dichlorophenoxyacetic acid (2,4-D), glyphosate (GF), dicamba, atrazine and metsulfuron-methyl] on the growth of rhizobial strains was assessed. Subsequently, GF and 2,4-D were tested in a minimum broth as C and energy sources for 20 tolerant strains. The ability of these strains to metabolize different carbon sources was studied in order to detect further differences among them. Tolerance of the bacteria to agrochemicals varied; 2,4-D and GF in solid medium inhibited and diminished growth, respectively, in slow-growing rhizobial strains. Among slow-growing strains we detected Bradyrhizobium sp. SJ140 that grew well in broth + GF as the sole C and energy source. No strain was found which could use 2,4-D as sole C source. The 20 strains studied exhibited different patterns of C sources utilization. Cluster analysis revealed three groups, corresponding to four genera of rhizobia: Rhizobium (group I), Sinorhizobium (group II) and Mesorhizobium–Bradyrhizobium (group III). On the basis of the results obtained on responses to herbicides and C sources utilization by the isolates investigated, it was possible to differentiate them at the level of strains. These results evidenced a considerable diversity in rhizobial populations that had not been previously described for Argentinean soils, and suggested a physiological potential to use natural and xenobiotic C sources.  相似文献   

18.
The compatibility between rhizobia and host plants for nodulation was determined based on the genetic and physiological properties of both symbionts. It has been observed that soybean varieties carrying the Rj-gene were not nodulated effectively by certain strains or groups of rhizobia. Soybeans carrying the Rj 2-gene, Rj 2-varieties, were found to nodule ineffectively by the rhizobial strains belonging to the 3-24-44 and 122 serogroups (Caldwell 1966). In the same way, Rj 3- and Rj 4-varieties were found to nodule ineffectively by strains USDA 33 (Vest 1970) and USDA 61 (Vest and Caldwell 1972), respectively.  相似文献   

19.
The nodulation of provenances of Acacia seyal, Acacia tortilis and Faidherbia albida, and other indigenous multipurpose tree species were tested in 14 different soil samples collected from diverse agro-ecological zones in southern Ethiopia. Associated rhizobia were isolated from these and from excavated nodules of field standing mature trees, and phenotypically characterized. Indigenous rhizobia capable of eliciting nodules on at least one or more of the woody legume species tested were present in most of the soils. Tree species were markedly different in nodulation in the different site soils. Sesbania sesban and Acacia abyssinica showed higher nodulation ability across the different sites indicating widespread occurrence of compatible rhizobia in the soils. The nodulation patterns of the different provenances of Acacia spp. suggested the existence of intraspecific provenance variations in rhizobial affinity which can be exploited to improve N fixation through tree selection. Altogether, 241 isolates were recovered from the root nodules of trap host species and from excavated nodules. Isolates were differentiated by growth rate and colony morphology and there were very fast-, fast-, slow-, and very slow-growing rhizobia. The bulk of them (68.5%) were fast-growing acid-producing rhizobia while 25.3% were slow-growing alkali-producing types. Fast-growing alkali-producing (2.9%) and slow-growing acid-producing strains (3.3%) were isolated from trap host species and excavated nodules, respectively. All isolates fell into four colony types: watery translucent, white translucent, dull glistering and milky (curdled) type. The diversity of indigenous rhizobia in growth rate and colony morphology suggested that the collection probably includes several rhizobial genera.  相似文献   

20.
The rhizobia-legume symbiosis is the main source of fixed nitrogen for many agricultural systems. However, it is inhibited by low soil temperature. To date, research on nodulation has involved either qualitative or destructive analyses. The use of computer-based image analysis potentially allows nodules to be followed during the course of development. Seedlings of bean (Phaseolus vulgaris L.), lentil (Lens culinaris Medik.) and pea (Pisum sativum L.) were transplanted into plastic growth pouches suspended in water baths maintained at 10, 15, 20 or 25 °C. Two days after transplanting, all plants were inoculated with appropriate rhizobial strains. Seven days after inoculation, plant roots were scanned; this was repeated weekly for 7 weeks. Data on nodule length were collected through image analysis. Nodule length was correlated with nodule size and development. There were increases in the precision of estimates of environmental effects through observation of individual nodule development, as opposed to averages for populations of nodules. The effects of root temperature on nodulation and nodule development were observed both in the delayed onset of nodulation and in reduced subsequent nodule growth rate, resulting in effects on final nodule size.  相似文献   

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