丛枝菌根真菌对红花根围微生物多样性特征的影响

[目的]研究丛枝菌根(Arbuscular mycorrhiza,AM)真菌对红花根围微生物多样性特征的影响.[方法]对红花接种Glomus mosseae、G.intraradices和混合菌(G.mosseae、G.intraradices、G.cladoideum、G.microagregatum、G.caledonium、G.etunicatum)3种AM真菌,采用传统的平板稀释涂布法对红花根围细菌、真菌、放线菌进行分离、培养、计数和鉴定,并结合单链构象多态性(Single stand conformational polymorphism,SSCP)分析技术对红花根围微生物多样性进行分析.[结果]3处理与对照组红花根围微生物总量表现为G.mosseae＞G.intraradices＞混合菌＞对照,且接种处理红花根围微生物总量显著高于对照.对照组红花各生长期微生物总量均为0-5 cm＞5 cm-10 cm＞10 cm-20 cm,接种处理红花生长至种子成熟期时不同土层微生物数量发生变化,微生物总量为5 cm-10 cm＞0-5 cm＞10 cm-20 cm.聚类分析发现微生物出现接种红花类群与不接种红花类群两大分类类群.[结论]AM真菌从时间和空间上影响了红花根围微生物的多样性特征,对红花农业生产和分析生态系统中微生物之间的相互作用具有重要的理论和实践价值.     

Greenhouse and Field Evaluations of an Autoselective System Based on an Essential Thymidylate Synthase Gene for Improved Maintenance of Plasmid Vectors in Modified Rhizobium meliloti

The stability of the thy autoselective system, based on an essential thymidylate synthase gene, for enhanced maintenance of plasmid vectors in Rhizobium meliloti was evaluated in the greenhouse and with field-grown alfalfa. The thy autoselective system consists of a free-replicating, broad-host-range plasmid vector containing a copy of the thyA gene from Lactococcus lactis subsp. lactis and a spontaneous mutant of R. meliloti deficient in thymidylate synthase (Thy(sup-)). Under greenhouse conditions, Thy(sup-) rhizobia did not persist in rooting solution alone unless supplemented with thymidine but survived in the presence of the host plant. Nodules formed on alfalfa plants grown in thymidine-free rooting solution and inoculated with Thy(sup-) rhizobia contained only Thy(sup+) revertants. In soil, Thy(sup-) rhizobia were compromised and failed to nodulate alfalfa. Thy(sup-) mutants containing a thy plasmid survived in the rhizosphere and nodulated alfalfa like the wild-type strain. The thy autoselective system was tested in the field with Thy(sup-) strain Rm24T and pPR602, a thy plasmid vector devoid of antibiotic resistance genes and marked with constitutively expressed lacZY. At 80 days after sowing, most rhizobia isolated from the nodules of field-grown alfalfa inoculated with Rm42T(pPR602) contained pPR602. The thy autoselective system proved useful to ensure maintenance of the plasmid vector under greenhouse and field conditions in R. meliloti.     

Interference between Rhizobium meliloti and Rhizobium trifolii nodulation genes: genetic basis of R. meliloti dominance.

Transfer of an IncP plasmid carrying the Rhizobium meliloti nodFE, nodG, and nodH genes to Rhizobium trifolii enabled R. trifolii to nodulate alfalfa (Medicago sativa), the normal host of R. meliloti. Using transposon Tn5-linked mutations and in vitro-constructed deletions of the R. meliloti nodFE, nodG, and nodH genes, we showed that R. meliloti nodH was required for R. trifolii to elicit both root hair curling and nodule initiation on alfalfa and that nodH, nodFE, and nodG were required for R. trifolii to elicit infection threads in alfalfa root hairs. Interestingly, the transfer of the R. meliloti nodFE, nodG, and nodH genes to R. trifolii prevented R. trifolii from infecting and nodulating its normal host, white clover (Trifolium repens). Experiments with the mutated R. meliloti nodH, nodF, nodE, and nodG genes demonstrated that nodH, nodF, nodE, and possibly nodG have an additive effect in blocking infection and nodulation of clover.     

Influence of arbuscular mycorrhizae and Rhizobium on nutrient content and water relations in drought stressed alfalfa

The objective of this research was to study the effect of drought on nutrient content and leaf water status in alfalfa (Medicago sativa L. cv Aragón) plants inoculated with a mycorrhizal fungus and/or Rhizobium compared with noninoculated ones. The four treatments were: a) plants inoculated with Glomus fasciculatum and Rhizobium meliloti 102 F51 strain, (MR); b) plants inoculated with R. meliloti only (R); c) plants with G. fasciculatum only (M); and d) noninoculated plants (N). Nonmycorrhizal plants were supplemented with phosphorus and nonnodulated ones with nitrogen to achieve similar size and nutrient content in all treatments. Plants were drought stressed using two cycles of moisture stress and recovery. The components of total leaf water potential (osmotic and pressure potentials at full turgor), percentage of apoplastic water volume and the bulk modulus of elasticity of leaf tissue were determined. Macronutrient (N, P, K, Ca, S and Mg) and micronutrient (Co, Mo, Zn, Mn, Cu, Na, Fe and B) content per plant were also measured. Leaves of N and R plants had decreased osmotic potentials and increased pressure potentials at full turgor, with no changes either in the bulk modulus of elasticity or the percentage of apoplastic water upon drought conditions. By contrast, M and MR leaves did not vary in osmotic and turgor potentials under drought stress but had increased apoplastic water volume and cell elasticity (lowering bulk modulus). Drought stress decreased nutrient content of leaves and roots of noninoculated plants. R plants showed a decrease in nutrient content of leaves but maintained some micronutrients in roots. Leaves of M plants were similar in content of nutrients to N plants. However, roots of M and MR plants had significantly lower nutrient content. Results indicate an enhancement of nutrient content in mycorrhizal alfalfa plants during drought that affected leaf water relations during drought stress.     

Requirement of succinate dehydrogenase activity for symbiotic bacteroid differentiation of Rhizobium meliloti in alfalfa nodules

Transmission electron microscopy was used to study the cellular morphologies of a wild-type Rhizobium meliloti strain (L5-30), a nitrogen fixation-ineffective (Fix-) succinate dehydrogenase mutant (Sdh-) strain, and a Fix+ Sdh+ revertant strain within alfalfa nodules and after free-living growth in a minimal medium containing 27 mM mannitol plus 20 mM succinate. The results showed a requirement of succinate dehydrogenase activity for symbiotic differentiation and maintenance of R. meliloti bacteroids within alfalfa nodules and for succinate-induced cellular pleomorphism in free-living cultures. Also, the Sdh- strain had a 3.5-fold lower rate of oxygen consumption in the defined medium than did the wild type.     

Molecular evidence of genetic modification of Sinorhizobium meliloti: enhanced PCB bioremediation

The genome of the nitrogen-fixing soil bacterium Sinorhizobium meliloti does not possess genes for bioremediation of aromatic pollutants. It has the well-known ability to interact specifically with the leguminous alfalfa plant, Medicago sativa. Our previous work has shown enhanced degradation of the nitroaromatic compound 2,4-dinitrotoluene (DNT) when a plasmid containing degradative genes was introduced in it. In this study we report molecular evidence of the transfer of a polychlorinated biphenyl (PCB)-biodegradative plasmid pE43 to S. meliloti strain USDA 1936. Several standard analytical tests and plant growth chamber studies were conducted to test the ability of S. meliloti to degrade 2',3,4-PCB congener. Alfalfa plant alone was able to degrade 30% of PCBs compared with control. No enhanced dechlorination was noted when alfalfa plant was grown with wild-type S. meliloti, and when alfalfa plant was grown with the S. meliloti electrotransformants (genetically modified) dechlorination of PCBs was more than twice that when alfalfa plant was grown with wild-type S. meliloti. When alfalfa plant was grown with uncharacterized mixed culture (containing nodule formers), almost equally significant PCB degradation was observed. The significance of this work is that the naturally occurring nitrogen-fixing soil bacterium S. meliloti (genetically modified) has the ability to enhance fertility of soil in association with the leguminous alfalfa plant while simultaneously enhancing bioremediation of PCB-contaminated soils. Enhanced bioremediation of PCB and robust alfalfa plant growth was also noted when uncharacterized mixed cultures containing alfalfa plant nodule formers were used.     

Requirement of succinate dehydrogenase activity for symbiotic bacteroid differentiation of Rhizobium meliloti in alfalfa nodules.

Transmission electron microscopy was used to study the cellular morphologies of a wild-type Rhizobium meliloti strain (L5-30), a nitrogen fixation-ineffective (Fix-) succinate dehydrogenase mutant (Sdh-) strain, and a Fix+ Sdh+ revertant strain within alfalfa nodules and after free-living growth in a minimal medium containing 27 mM mannitol plus 20 mM succinate. The results showed a requirement of succinate dehydrogenase activity for symbiotic differentiation and maintenance of R. meliloti bacteroids within alfalfa nodules and for succinate-induced cellular pleomorphism in free-living cultures. Also, the Sdh- strain had a 3.5-fold lower rate of oxygen consumption in the defined medium than did the wild type.     

Sugar-binding activity of pea lectin enhances heterologous infection of transgenic alfalfa plants by Rhizobium leguminosarum biovar viciae

Transgenic alfalfa (Medicago sativa L. cv Regen) roots carrying genes encoding soybean lectin or pea (Pisum sativum) seed lectin (PSL) were inoculated with Bradyrhizobium japonicum or Rhizobium leguminosarum bv viciae, respectively, and their responses were compared with those of comparably inoculated control plants. We found that nodule-like structures formed on alfalfa roots only when the rhizobial strains produced Nod factor from the alfalfa-nodulating strain, Sinorhizobium meliloti. Uninfected nodule-like structures developed on the soybean lectin-transgenic plant roots at very low inoculum concentrations, but bona fide infection threads were not detected even when B. japonicum produced the appropriate S. meliloti Nod factor. In contrast, the PSL-transgenic plants were not only well nodulated but also exhibited infection thread formation in response to R. leguminosarum bv viciae, but only when the bacteria expressed the complete set of S. meliloti nod genes. A few nodules from the PSL-transgenic plant roots were even found to be colonized by R. leguminosarum bv viciae expressing S. meliloti nod genes, but the plants were yellow and senescent, indicating that nitrogen fixation did not take place. Exopolysaccharide appears to be absolutely required for both nodule development and infection thread formation because neither occurred in PSL-transgenic plant roots following inoculation with an Exo(-) R. leguminosarum bv viciae strain that produced S. meliloti Nod factor.     

Inoculation of field-established mulberry and papaya with arbuscular mycorrhizal fungi and a mycorrhiza helper bacterium

The effects of soil inoculation with arbuscular mycorrhizal (AM) fungi and a mycorrhiza helper bacterium (MHB) were investigated on mulberry and papaya plants already established in the field. Ten-year-old mulberry plants (var. M5) were inoculated with Glomus fasciculatum and 1.5-year-old papaya plants (var. Solo) were inoculated with a mixed culture of G. mosseae and G. caledonium with or without Bacillus coagulans at two levels of P fertilizer. Growth, P uptake, yield and AM colonization levels were monitored. Leaf yield in mulberry and fruit yield in papaya were minimal in uninoculated plants given 50% recommended P. However, crop yields of both mulberry and papaya inoculated with AM fungi alone or together with the bacterium and given 50% recommended P were statistically on a par with that of uninoculated plants given 100% recommended P. As inoculation of B. coagulans increased mycorrhiza levels in AM fungal-inoculated plants, this may be included in the class of MHB. Thus, mulberry and papaya already established in the field may respond to AM inoculation and MHB may increase symbiosis development by efficient AM fungi.     

Transconjugants of Agrobacterium radiobacter harbouring sym genes of Rhizobium galegae can form an effective symbiosis with Medicago sativa

It is known that the Rhizobium galegae genomes contain megaplasmids. The suicide vector pSUP2111 with nifH gene of R. meliloti was introduced into the strains CIAM 0703 and CIAM 0711 of R. galegae inducing effective nodules on Galega orientalis plants. The formation of self-transmissible megaplasmids was observed. The megaplasmid transfer into non-nodulating R. meliloti mutants resulted in partial complementation of the nodulation defect in recipient strains though only one transconjugant showed the nitrogen-fixing activity in symbiosis with alfalfa and another one in symbiosis with G. orientalis plants. Among the Agrobacterium strains harbouring R. galegae megaplasmids there were four classes of transconjugants: (1) Nod+ Fix- in symbiosis with goat's rue plants (three strains); (2) Nod+ Fix- on Medicago sativa (two strains); (3) Nod+ Fix+ on M. sativa (five strains); (4) Nod- with both plant hosts (11 strains).     

Rhizobium meliloti mutants unable to synthesize anthranilate display a novel symbiotic phenotype.

Analyses of Rhizobium meliloti trp auxotrophs suggest that anthranilate biosynthesis by the R. meliloti trpE(G) gene product is necessary during nodule development for establishment of an effective symbiosis. trpE(G) mutants, as well as mutants blocked earlier along this pathway in aromatic amino acid biosynthesis, form nodules on alfalfa that have novel defects. In contrast, R. meliloti trp mutants blocked later in the tryptophan-biosynthetic pathway form normal, pink, nitrogen-fixing nodules. trpE(G) mutants form two types of elongated, defective nodules containing unusually extended invasion zones on alfalfa. One type contains bacteroids in its base and is capable of nitrogen fixation, while the other lacks bacteroids and cannot fix nitrogen. The trpE(G) gene is expressed in normal nodules. Models are discussed to account for these observations, including one in which anthranilate is postulated to act as an in planta siderophore.     

The symbiotic defect of Rhizobium meliloti exopolysaccharide mutants is suppressed by lpsZ+, a gene involved in lipopolysaccharide biosynthesis.

exo mutants of Rhizobium meliloti SU47, which fail to secrete acidic extracellular polysaccharide (EPS), induce Fix- nodules on alfalfa. However, mutants of R. meliloti Rm41 carrying the same exo lesions induce normal Fix+ nodules. We show that such induction is due to a gene from strain Rm41, which we call lpsZ+, that is missing in strain SU47. lpsZ+ does not restore EPS production but instead alters the composition and structure of lipopolysaccharide. In both SU47 and Rm41, either lpsZ+ or exo+ is sufficient for normal nodulation. This suggests that in R. meliloti EPS and lipopolysaccharide can perform the same function in nodule development.     

Early recognition in the Rhizobium meliloti-alfalfa symbiosis: root exudate factor stimulates root adsorption of homologous rhizobia.

Adsorption of Rhizobium meliloti to alfalfa roots before their infection and nodule formation shows the specificity of the symbiotic association (G. Caetano-Anollés and G. Favelukes, Appl. Environ. Microbiol. 52:377-382, 1986). The time course of specific adsorption of R. meliloti (10(3) to 10(4) cells per ml) to roots shows an initial lag period of 3 h, suggesting that either or both symbionts must become conditioned for the adsorption process. Preincubation of R. meliloti L5-30 for 3 h with dialyzed alfalfa root exudate (RE) markedly increased early adsorption of rhizobia to alfalfa roots. The activity in RE was linked to a nondialyzable, thermolabile, trypsin-sensitive factor(s), very different from the root-exuded flavonoid compounds also involved in early Rhizobium-legume interactions. The lack of activity in the RE from plants grown in 5 mM NO3- suggested its negative regulation by the nitrogen nutritional status of the plant. Preincubation of R. meliloti with heterologous clover RE did not stimulate adsorption of rhizobial cells to roots. A short pretreatment of RE with homologous (but not heterologous) strains eliminated the stimulatory activity from solution. The stimulation of adsorption of R. meliloti to alfalfa roots was strongly dependent on the growth phase of the rhizobia, being greater at the late exponential stage. Nevertheless, the capacity of R. meliloti L5-30 to eliminate from solution the stimulatory activity in RE appeared to be constitutive in the rhizobia. The low concentration of rhizobial cells used in these experiments was critical to detect the stimulation of adsorption. The early interaction of spontaneously released alfalfa root macromolecular factor(s) and free-living R. meliloti, which shows the specificity and regulatory properties characteristic of infection and nodulation, would be an initial recognition event in the rhizosphere which triggers the process of symbiotic association.     

Potential of Phytoremediation for Treatment of PAHs in Soil at MGP Sites

Phytoremediation is a natural, aesthetically pleasing, low-cost technology that employs plant-influenced microbial, chemical, and physical processes to remediate contaminated soils and waters. The Institute of Gas Technology (IGT) conducted a laboratory study to determine the potential of phytoremediation to remediate soils contaminated with polynuclear aromatic hydrocarbons (PAHs). The soils used for the study were collected from a former manufactured gas plant (MGP) site in Newark, NJ. Phytoremediation was assessed both as a primary remediation technology and as a final polishing step for soil treatment. The following three plant species were used for the 6-month laboratory study: alfalfa (Medicago sativa), switch grass (Panicum virgatum), and little bluestem grass (Schizachyrium scoparium). Using both alfalfa and switch grass for primary treatment of PAH-contaminated soil, a 57% reduction in total PAH concentration was observed after 6-months of treatment. Final polishing of that soil using alfalfa further reduced the total PAH concentration in that soil by 15%. Research is in progress with the objective of improving both the efficiency and the economics of phytoremediation for the cleanup of contaminated soils to environmentally acceptable endpoints at MGP sites.     

Phylogenetic position of Rhizobium sp. strain Or 191, a symbiont of both Medicago sativa and Phaseolus vulgaris, based on partial sequences of the 16S rRNA and nifH genes.

Phenotypic and DNA sequence comparisons are presented for eight Rhizobium isolates that were cultured from field-grown alfalfa (Medicago sativa L.) in Oregon. These isolates were previously shown to nodulate both alfalfa and common bean (Phaseolus vulgaris (L.) Savi.). The objective of the present study was to determine their phylogenetic relationships to the normal symbionts of these plants, Rhizobium meliloti and Rhizobium leguminosarum biovar phaseoli, respectively. Phenotypically, the Oregon isolates more nearly resemble strains from P. vulgaris than those from M. sativa. For example, even though nitrogen fixation levels were low with both host species, the symbiotic efficiency of a representative Rhizobium isolate (Or 191) with common bean was twice that observed with alfalfa. Comparative sequencing of a 260-bp segment of the 16S rRNA gene (directly sequenced after amplification by the polymerase chain reaction) demonstrated that Or 191 is not closely related to the type strain of R. meliloti (ATCC 9930), R. leguminosarum (ATCC 10004), or Rhizobium tropici (CIAT 899). Instead, sequence comparisons of the 16S gene indicated that Or 191 belongs to a distinct and previously unrecognized taxonomic group that includes strains that have previously been called R. leguminosarum bv. phaseoli type I. Unlike type I strains, however, Or 191 has only a single copy of the nifH gene (type I strains have three), and the nucleotide sequence of this gene is substantially different from those of other rhizobial and nonrhizobial nifH genes examined thus far.     

Phylogenetic position of Rhizobium sp. strain Or 191, a symbiont of both Medicago sativa and Phaseolus vulgaris, based on partial sequences of the 16S rRNA and nifH genes.

Phenotypic and DNA sequence comparisons are presented for eight Rhizobium isolates that were cultured from field-grown alfalfa (Medicago sativa L.) in Oregon. These isolates were previously shown to nodulate both alfalfa and common bean (Phaseolus vulgaris (L.) Savi.). The objective of the present study was to determine their phylogenetic relationships to the normal symbionts of these plants, Rhizobium meliloti and Rhizobium leguminosarum biovar phaseoli, respectively. Phenotypically, the Oregon isolates more nearly resemble strains from P. vulgaris than those from M. sativa. For example, even though nitrogen fixation levels were low with both host species, the symbiotic efficiency of a representative Rhizobium isolate (Or 191) with common bean was twice that observed with alfalfa. Comparative sequencing of a 260-bp segment of the 16S rRNA gene (directly sequenced after amplification by the polymerase chain reaction) demonstrated that Or 191 is not closely related to the type strain of R. meliloti (ATCC 9930), R. leguminosarum (ATCC 10004), or Rhizobium tropici (CIAT 899). Instead, sequence comparisons of the 16S gene indicated that Or 191 belongs to a distinct and previously unrecognized taxonomic group that includes strains that have previously been called R. leguminosarum bv. phaseoli type I. Unlike type I strains, however, Or 191 has only a single copy of the nifH gene (type I strains have three), and the nucleotide sequence of this gene is substantially different from those of other rhizobial and nonrhizobial nifH genes examined thus far.     

Legume agglutinins that bind to Rhizobium meliloti.

A protein found in seeds and roots of alfalfa (Medicago sativa) was implicated in the specificity of the infection process, based on its binding to the symbiont Rhizobium meliloti. We found an agglutinin with similar properties in seeds and roots of sweet clover (Melilotis alba). The sweet clover differed from alfalfa in nodulation by a mutant strain of R. meliloti, but the agglutinins were indistinguishable by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Rhizobium agglutination, and cross-reactivity to antibodies. Similar agglutinins binding R. meliloti were found in seeds of legumes from different cross-inoculation groups, including soybean (Glycine max), cowpea (Vigna unguiculata), pea (Pisum sativum L), and mung bean (Vigna mungo). The agglutinins from these legumes were recognized by antibodies raised against the agglutinins of alfalfa and sweet clover. Seeds of corn (Zea mays) and tomato (Lycopersicon esculentum) contained a protein similar to the legume agglutinin, but it did not react with the antibodies. We conclude that the alfalfa agglutinin is representative of a common legume protein and that there is no evidence for its role in specificity or nodule initiation.     

Bacterial Growth Rates and Competition Affect Nodulation and Root Colonization by Rhizobium meliloti

The addition of streptomycin to nonsterile soil suppressed the numbers of bacterial cells in the rhizosphere of alfalfa (Medicago sativa L.) for several days, resulted in the enhanced growth of a streptomycin-resistant strain of Rhizobium meliloti, and increased the numbers of nodules on the alfalfa roots. A bacterial mixture inoculated into sterile soil inhibited the colonization of alfalfa roots by R. meliloti, caused a diminution in the number of nodules, and reduced plant growth. Enterobacter aerogenes, Pseudomonas marginalis, Acinetobacter sp., and Klebsiella pneumoniae suppressed the colonization by R. meliloti of roots grown on agar and reduced nodulation by R. meliloti, the suppression of nodulation being statistically significant for the first three species. Bradyrhizobium sp. and “Sarcina lutea” did not suppress root colonization nor nodulation by R. meliloti. The doubling times in the rhizosphere for E. aerogenes, P. marginalis, Acinetobacter sp., and K. pneumoniae were less and the doubling times for Bradyrhizobium sp. and “S. lutea” were greater than the doubling time of R. meliloti. Under the same conditions, Arthrobacter citreus injured alfalfa roots. We suggest that competition by soil bacteria reduces nodulation by rhizobia in soil and that the extent of inhibition is related to the growth rates of the rhizosphere bacteria.     

Effect of arbuscular mycorrhizal fungal inoculation on heavy metal accumulation of maize grown in a naturally contaminated soil

A pot culture experiment was carried out to study heavy metal (HM) phytoaccumulation from soil contaminated with Cu, Zn, Pb, and Cd by maize (Zea mays L.) inoculated with arbuscular mycorrhizal (AM) fungi (AMF). Two AM fungal inocula--MI containing only one AM fungal strain (Glomus caledonium 90036) and MII consisting of Gigaspora margarita ZJ37, Gigaspora decipens ZJ38, Scutellospora gilmori ZJ39, Acaulospora spp., and Glomus spp.--were applied to the soil under unsterilized conditions. The control received no mycorrhizal inoculation. The maize plants were harvested after 10 wk of growth. MI-treated plants had higher mycorrhizal colonization than MII-treated plants. Both MI and MII increased P concentrations in roots, but not in shoots. Neither MI nor MII had significant effects on shoot or root dry weight (DW). Compared with the control, shoot Cu, Zn, Pb, and Cd concentrations were decreased by MI but increased by MII. Cu, Zn, Pb, and Cd uptake into shoots and roots all increased in MII-treated plants, while in MI-treated plants Cu, Zn, and Pb uptake into shoots and Cd uptake into roots decreased but Cu, Zn, and Pb uptake into roots and Cd into shoots increased. MII was more effective than MI in promoting HM extraction efficiencies. The results indicate that MII can benefit HMphytoextraction and, therefore, show potential in the phytoremediation of HM-contaminated soils.