Factors affecting co-inoculation with antibiotic-producing bacteria to enhance rhizobial colonization and nodulation

Co-inoculation with antibiotic-producing bacteria and rhizobia resistant to those antibiotics has been proposed as a means of promoting colonization and nodulation of legumes by root-nodule bacteria. A study was conducted to establish some of the factors affecting co-inoculation with antibiotic-producing strains of Bacillus and Streptomyces griseus. The stimulation of Rhizobium meliloti and yield and N uptake by alfalfa was enhanced with increasing inoculum size of Bacillus sp. S. griseus and chitin added to soil increased nodulation of soybeans by Bradyrhizobium japonicum and increased nodulation, yield, and number of pods on a second crop grown in the same soil. Bacillus sp. persisted in soil in sufficient numbers for at least 51 days to increase colonization of soybean roots by B. japonicum. The populations of S. griseus, Bacillus sp., and antibiotic-resistant isolates of R. meliloti and B. japonicum fell after their addition to seeds. Nevertheless, a benefical effect by the antibiotic-producing bacteria was evident on R. meliloti colonization of the rhizosphere, nodulation, and yield of alfalfa grown from seeds stored 94 days and on B. japonicum colonization, nodule number, yield, and seed weight of soybeans grown from seeds stored 90 days. Because non-antibiotic-producing derivatives of Bacillus sp. and S. griseus did not promote colonization or nodulation of alfalfa roots by R. meliloti, the benefit of this co-inoculation is a result of antibiotic formation.     

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.     

The Biological Activity of the Sinorhizobium meliloti Glucan

The study of the effect of periplasmic glucan isolated from the root-nodule bacterium Sinorhizobium meliloti CXM1-188 on the symbiosis of another strain (441) of the same root-nodule bacterium with alfalfa plants showed that this effect depends on the treatment procedure. The pretreatment of alfalfa seedlings with glucan followed by their bacterization with S. meliloti 441 insignificantly influenced the nodulation parameters of symbiosis (the number of root nodules and their nitrogen-fixing activity) but induced a statistically significant increase in the efficiency of symbiosis (expressed as the masses of the alfalfa overground parts and roots). At the same time, the pretreatment of S. meliloti 441 cells with glucan brought about a considerable decrease in the nodulation parameters of symbiosis (the number of root nodules and their nitrogen-fixing activity decreased by 2.5–11 and 7 times, respectively). These data suggest that the stimulating effect of rhizobia on host plants may be due not only to symbiotrophic nitrogen fixation but also to other factors. Depending on the experimental conditions, the treatment of alfalfa plants with glucan and their bacterization with rhizobial cells enhanced the activity of peroxidase in the alfalfa roots and leaves by 10–39 and 12–27%, respectively.     

A new perfusion system for the measurement and characterization of potential rates of soil nitrification

The effect of two isoflavonoids, coumestrol and daidzein which are present in aseptically grown roots and root exudates of soybean, was tested on some rhizospheric microorganisms. It was found that coumestrol promotes the growth ofR. japonicum USDA 138 (about 30%) andR. leguminosarum (about 15%) whereas it inhibits the growth ofAgrobacterium tumefaciens (about 50%) andPseudomonas sp. (about 20%). The following microorganisms were unaffected by this molecule:R. japonicum W505,Agrobacterium radiobacter, Micrococcus luteus andCryptococcus laurentii. It was found that daidzein promotesR. japonicum USDA 138 growth (about 20%) and inhibitsPseudomonas sp. growth (about 20%); other microorganisms were unaffected. In addition, coumestrol favoured the formation of ‘coccoids’ cells byRhizobium japonicum USDA 138 which could be the infective state of this strain. It seems that this compound is able to help nodulation of soybean by aRhizobium strain. This result supports the work of Peterset al. (1986) and Redmondet al. (1986) who show that flavones present in plant exudates induces expression of nodulation genes in Rhizobium.     

Effects of mycorrhizae and chitin-hydrolysing microbes on Vicia faba

The effect of Streptomyces albovinaceus (S-22) and Bacillus sp. (B1) on the growth response, nodulation, nutrition and nitrogenase activities of faba bean (Vicia faba) varieties infected with Glomus mosseae under pot conditions in sterile soil amended with chitin was studied. The growth, nodulation, nutrients content and nitrogenase activity of mycorrhiza-treated plants of Giza-667 were significantly increased compared to untreated ones. Such increases were related to the increase in mycorrhizal root infection. Amendment of soil with chitin alone reduced the growth, nodulation, total nitrogen contents and nitrogenase activities of mycorrhiza-treated faba bean plants (Giza-667) compared to untreated plants. Inoculation of plants with S. albovinaceus or Bacillus sp. significantly increased the level of mycorrhizal roots infection, but addition of chitin to the soil in combination with Bacillus sp. reduced the mycorrhizal infection of faba bean roots. Highest phosphorus contents of faba bean Giza-667 were recorded after G. mosseae inoculation in the presence of all treatments. Similar results were observed for the other varieties. The microbial populations were significantly increased in rhizospheres amended with chitin. Such increases were not in response to the mycorrhizal inoculation. Generally, the microflora of faba bean rhizospheres was increased after treatment with G. mosseae in the absence of chitin amendment alone compared with non-mycorrhizal rhizospheres.     

Antigenic affinities of the root-nodule bacteria of legumes

Antisera prepared against 58 strains of root-nodule bacteria and against 16 strains belonging to the genusAgrobacterium were tested against 113 strains ofRhizobium, 20 strains ofAgrobacterium and 20 strains of other, possibly related, bacteria.Three serologically distinct groups of root-nodule bacteria were noted: (1)Rh. trifolii, Rh. leguminosarum andRh. phaseoli; (2)Rh. lupini, Rh. japonicum andRhizobium spp.; (3)Rh. meliloti. Strains ofRh. meliloti showed serological affinities withA. radiobacter andA. tumefaciens. All groups showed wider flagellar than somatic agglutination, and many different serotypes were apparent.The groupings obtained from this investigation are compared with those derived from other taxonomic studies, and the use of serological methods in rhizobial classification is discussed.     

Colonization of cotton roots by soil micro-organisms

An experiment was carried out to study the colonization of soil microorganisms on cotton roots buried in the soil.Fusarium culmorum, F. solani, F. oxysporum, Macrophomina phaseoli andBacillus sp. were found to be the predominant colonizers followed byBotryodiplodia theobromae, Fusarium acuminatum, Aspergillus niger, A. flavus, Stachybotrys atra, Tilachlidium humicola andRhizopus sp. The practice of leaving cotton roots in the soil would therefore add to the building up of the inoculum potential of the soil inhabiting fungi several of which are serious root pathogens.     

Rhizobium meliloti inoculation of alfalfa selected for tolerance to acid,aluminum-rich soils

Alfalfa (Medicago sativa L.) growth and nodulation in acid soil is reduced because the plant and its bacterial symbiontRhizobium meliloti cannot tolerate acid, aluminum-rich soil. A study was conducted to determine if a relatively acid-tolerant alfalfa germplasm combined with a relatively acid-tolerantR. meliloti strain could overcome these limitations. In a light room study, an acid-tolerant alfalfa germplasm inoculated with a more acid-tolerantR. meliloti strain produced greater top growth, nodule number and weight, and acetylene reduction values in an unlimed soil (pH 4.6) than the same germplasm inoculated with a relatively acid-sensitiveR. meliloti strain or an acid-sensitive germplasm inoculated with either a relatively acid-tolerant or acid-sensitiveR. meliloti strain.     

DNA-DNA hybridization of some root-nodule bacteria indigenous in the salt-affected soils of egypt

The DNA-DNA hybridization was used to characterize thirty isolates of root-nodule bacteria indigenous to the salt-affected soils of Egypt. Total DNA from different bacterial isolates lacked homology with total DNA probes of the effective strains ofRhizobium leguminosarum andR. meliloti. It is suggested that the genomic structure of the root-nodule bacteria may be modified by salt stress and/or that the effective strains of these bacteria are to be eliminated from the salt-affected soil.     

Relative genetic structure of a population of Rhizobium meliloti isolated directly from soil and from nodules of alfalfa (Medicago sativa) and sweet clover (Melilotus alba)

Insertion sequence (IS) hybridization was used to define the structure of a population of Rhizobium meliloti isolated directly from soil and from nodules of Medicago sativa (alfalfa) and Melilotus alba (sweet clover) grown under controlled conditions and inoculated with a suspension of the same soil. The detection of R. meliloti isolated from soil on agar plates was facilitated by use of a highly species specific DNA probe derived from ISRm5. All R. meliloti obtained directly from soil proved to be symbiotic (i.e. nodulated and fixed nitrogen with alfalfa). Analysis of 293 R. meliloti isolates revealed a total of 17 distinct IS genotypes of which 9, 9 and 15 were from soil, M. alba and M. sativa, respectively; 8 genotypes were common to soil and both plant species. The frequency of R. meliloti genotypes from soil differed markedly from that sampled from nodules of both legume species: 5 genotypes represented about 90% of the isolates from soil whereas a single genotype predominated among isolates from nodules accounting for more than 55% of the total. The distribution of genotypes differed between M. sativa and M. alba indicating species variation in nodulation preferences for indigenous R. meliloti. The data are discussed in the context of competition for nodulation of the host plant and the selection of Rhizobium strains for use in legume inoculants. This study has ecological implications and suggests that the composition of R. meliloti populations sampled by the traditionally used host legume may not be representative of that actually present in soil.     

Attachment,colonization and proliferation ofAzospirillum brasilense andEnterobacter spp. on root surface of grasses

Root colonization studies, employing immunofluorescence and using locally isolated strains, showed thatEnterbacter sp. QH7 andEnterobacter agglomerans AX12 attached more readily to the roots of most plants compared withAzospirillum brasilense JM82. Heat treatment of either root or inoculum significantly decreased the adsorption of bacteria to the root surface. Kallar grass and rice root exudates sustained the growth ofA. brasilense JM82,Enterobacter sp. QH7 andE. agglomerans AX12 in Hoagland and Fahraeus medium. All the strains colonized kallar grass and rice roots in an axenic culture system. However, in studies involving mixed cultures,A. brasilense JM82 was inhibited byEnterobacter sp. QH7 in kallar grass rhizosphere and the simultaneous presence ofEnterobacter sp. QH7 andE. agglomerans AX12 suppressed the growth ofA. brasilense JM82 in rice rhizosphere. The bacterial colonization pattern changed from dispersed to aggregated within 3 days of inoculation. The colonization sites corresponded mainly to the areas where root mucigel was present. The area around the point of emergence of lateral roots usually showed maximum colonization.     

Positive role of nodulation on the establishment ofRhizobium japonicum in subsequent crops of soybean

The influence of soybean nodulation on the establishment ofRhizobium japonicum inRhizobium-free soil was examined. Seeds of nodulating (Rj ₁) and nonnodulating (rj ₁) isolines of soybeans and four other crop species (cowpeas, mungbeans, corn, and alfalfa) were grown in field plots that were inoculated with a genetically marked strain ofRhizobium (strain I-110 ARS) and the following year nodulating soybeans were grown in these plots and were inoculated with a different genetically marked subline of the same strain (strain I-110 FN). The proportion of nodules containing strain I-110 ARS relative to strain I-110 FN was determined and interpreted as reflecting the relative numbers of the two genetically marked sublines in the soil. The results clearly demonstrate that nodulation with the specific host plant (soybeans) has a significant positive role in the establishment ofRhizobium inRhizobium-free soil and suggests that alfalfa plants diminish the establishment of soybean rhizobia in soil.     

Enhanced nodule initiation on alfalfa by wild-typeRhizobium meliloti co-inoculated withnod gene mutants and other bacteria

Nodule formation on alfalfa (Medicago sativa L.) roots was determined at different inoculum dosages for wild-typeRhizobium meliloti strain RCR2011 and for various mutant derivatives with altered nodulation behavior. The number of nodules formed on the whole length of the primary roots was essentially constant regardless of initial inoculum dosage or subsequent bacterial multiplication, indicative of homeostatic regulation of total nodule number. In contrast, the number of nodules formed in just the initially susceptible region of these roots was sigmoidally dependent on the number of wild-type bacteria added, increasing rapidly at dosages above 5·10³ bacteria/plant. This behavior indicates the possible existence of a threshold barrier to nodule initiation in the host which the bacteria must overcome. When low dosages of the parent (10³ cells/plant) were co-inoculated with 10⁶ cells/plant of mutants lacking functionalnodA, nodC, nodE, nodF ornodH genes, nodule initiation was increased 10- to 30-fold. Analysis of nodule occupancy indicated that these mutants were able to help the parent (wild-type) strain initiate nodules without themselves occupying the nodules. Co-inoculation withR. trifolii orAgrobacterium tumefaciens cured of its Ti plasmid also markedly stimulated nodule initiation by theR. meliloti parent strain. Introduction of a segment of the symbiotic megaplasmid fromR. meliloti intoA. tumefaciens abolished this stimulation.Bradyrhizobium japonicum and a chromosomal Tn5 nod^- mutant ofR. meliloti did not significantly stimulate nodule initiation when co-inoculated with wild-typeR. meliloti. These results indicate that certainnod gene mutants and members of theRhizobiaceae may produce extracellular signals that supplement the ability of wild-typeR. meliloti cells to induce crucial responses in the host.Abbreviations EH emergent root hairs - kb kilobase - RDU relative distance unit - RT root tip This is journal article No. 188-87 of the Ohio Agricultural Research and Development Center     

Soybean biofertilization in sandy soils of Egypt using15N tracer technique

The effect of inoculating soybean plants withBradyrhizobium japonicum andAzospirillum brasilense either solely or in mixture, and of using different N fertilizer levels was studied in pot experiments. The nodulation of soybean grown in a sandy soil was enhanced by the inoculation while the highest nodule numbers and fresh mass, recorded at a N-fertilizer dose of 20 kg N/hm², decreased when the fertilizer quantity increased to 40 kg N/hm². By contrast, the dry mass of above-ground parts and the N uptake was increased with increasing N fertilizer level. A similar effect was observed for inoculation as compared with the uninoculated variant. Although the nodulating and non-nodulating soybeans has nearly the same dry mass, the nodulating isoline accumulated more N than the non-nodulating. The percentage of nitrogen derived from air (%, Ndfa) and estimated by isotope dilution (ID) or N difference method (DM) dropped with increasing N level from 10 to 40 kg N/hm². Dual inoculation resulted in a high per cent of fixed N₂ (42.5%) at 10 kg N/hm². Correlation between the ID and DM methods was found to be dependent on inoculation treatments. The amount of nitrogen utilized by nodulating soybean (FUE %) was enhanced as a function of inoculation withB. japonicum.     

Synthesis,release, and transmission of alfalfa signals to rhizobial symbionts

In addition to the flavonoids exuded by many legumes as signals to their rhizobial symbionts, alfalfa (Medicago sativa L.) releases two betaines, trigonelline and stachydrine, that induce nodulation (nod) genes inRhizobium meliloti. Experiments with¹⁴C-phenylalanine in the presence and absence of phenylalanine ammonia-lyase inhibitors show that exudation of flavonoidnod-gene inducers from alfalfa roots is linked closely to their concurrent synthesis. In contrast, flavonoid and betainenod-gene inducers are already present on mature seeds before they are released during germination. Alfalfa seeds and roots release structurally differentnod-gene-inducing signals in the absence of rhizobia. WhenR. meliloti is added to roots, medicarpin, a classical isoflavonoid phytoalexin normally elicited by pathogens, and anod-gene-inducing compound, formononetin-7-O-(6-O-malonylglycoside), are exuded. Carbon flow through the phenylpropanoid pathway and into the flavonoid pathway via chalcone synthase is controlled by complexcis-acting sequences andtrans-acting factors which are not completely understood. Even less information is available on molecular regulation of the two other biosynthetic pathways that produce trigonelline and stachydrine. Presumably the three separate pathways for producingnod-gene inducers in some way protect the plant against fluctuations in the production or transmission of the two classes of signals. Factors influencing transmission of alfalfanod-gene inducers through soil are poorly defined, but solubility differences between hydrophobic flavonoids and hydrophilic betaines suggest that the diffusional traits of these molecules are not similar. Knowledge derived from studies of how legumes regulate rhizobial symbionts with natural plant products offers a basis for defining new fundamental concepts of rhizosphere ecology.     

Interaction of vesicular arbuscular mycorrhiza with root knot nematodes in tomato

Summary The interaction between the VA mycorrhizal fungus,Glomus fasciculatus and the root-knot nematodes,Meloidogyne incognita andM. javanica, and their effects on the growth and phosphorus nutrition of tomato was studied in a red sandy loam soil of pH 6.0. Inoculation of tomato roots with root-knot nematodes enhanced infection and spore production byG. fasciculatus. Inoculation of tomato plants withG. fasciculatus significantly reduced the number and size of the root-knot galls produced byM. incognita andM. javanica. Inoculation withG. fasciculatus although improved plant growth and its total phosphorus content compared to the uninoculated plants, the difference were not statistically significant.     

Receptor Site on Clover and Alfalfa Roots for Rhizobium

Sites on white clover and alfalfa roots that bind Rhizobium trifolii and R. meliloti capsular polysaccharides, respectively, were examined by fluorescence microscopy. Fluorescein isothiocyanate-labeled capsular material from R. trifolii bound specifically to root hairs of clover but not alfalfa. Binding was most intense at the root hair tips. Treatment of clover roots with 2-deoxyglucose (2-dG) prevented binding of R. trifolii capsular material to the roots. The sugar 2-dG enhanced the elution of clover root protein, which could bind to and specifically agglutinate R. trifolii but not R. meliloti or R. japonicum. The mild elution procedure left the roots intact. Agglutination of R. trifolii and passive hemagglutination of rabbit erythrocytes coated with the capsular material of R. trifolii were specifically inhibited by 2-dG. These results suggest that clover roots contain proteins that cross-link complementary polysaccharides on the surface of clover root hairs and infective R. trifolii through 2-dG-sensitive binding sites. Alfalfa root hairs were shown to specifically bind to a surface polysaccharide from R. meliloti.     

Influence of the herbicide phosphinothricin on growth and nodulation capacity of Rhizobium meliloti

  Rhizobium meliloti proved to be sensitive to low concentrations of the herbicide phosphinothricintripeptide (PTT) and its active ingredient phosphinothricin (PT), which was formerly assumed to be non-toxic for most of the bacteria analysed. Growth was more strongly reduced in sterile synthetic media and less reduced in sterile soil; in unsterile soil only a transient growth reduction was detectable. Sensitivity was also observed in five out of eight other species analysed. In all sensitive species tested, spontaneous resistances to PT occurred. Under sterile conditions, PTT and PT reduced rhizobial nodulation rates of PT-resistant alfalfa plants drastically; however, nitrogen fixation in the few nodules that arose was unaffected. Because of the small number of nodules, the overall fixation rate was strongly diminished. In unsterile soil, nodulation and nitrogen fixation rates were not changed, possibly because of the rapid degradation of PTT and PT in the soil. Using a herbicide as model substance it could be demonstrated that the sensitivity of R. meliloti to chemical additives in the soil can be detected by analysing its growth rate, and that even a weak impact can influence its nodulation capacity. R. meliloti has proven to be a fast, easy and sensitive detection system for bacteriostatic components present in the soil. Received: 12 April 1996 / Received revision: 15 July 1996 / Accepted: 18 July 1996     

Influence of sodium humate on the crop plants inoculated with bacteria of agricultural importance

Summary The dry-matter yield and nitrogen uptake of berseem (Trifolium alexand-drinum), yield, nitrogen uptake, nodulation and leghaemoglobin content of dhaincha (Sesbania aculeata) inoculated with specific rhizobia were appreciably influenced by the application of sodium humate to soil under green house conditions. Even the application of sodium humate alone without bacterial inoculation had good growth stimulating influence on both the crops, and this effect was further improved by the application of inorganic nitrogen to dhaincha plants. A fair increase in the yield and phosphorus up-take of wheat (Triticum vulgare) inoculated withAzotobacter and/orBacillus spp. was also recorded with the addition of the humic material to the soil. The greatest effect was observed on the plants inoculated withAzotobacter andBacillus spp. together.     

Dual inoculation with VA mycorrhiza and Rhizobium is beneficial to Leucaena

Summary Response ofLeucanea leucocephala to inoculation withGlomus fasciculatum and/or Rhizobium was studied in a phosphorus deficient unsterile soil.G. fasciculatum only inoculation improved nodulation by native rhizobia and Rhizobium only treatment improved colonization of roots by native mycorrhizal fungi. Dual inoculation with both the organisms improved nodulation, mycorrhizal colonization, dry weight, nitrogen and phosphorus content of the plants compared to single inoculation with either organism. Contribution of U.A.S. Research Project DR/AMB-1.