Evidence Suggesting Protozoan Predation on Rhizobium Associated with Germinating Seeds and in the Rhizosphere of Beans (Phaseolus vulgaris L.)

Changes in populations of microorganisms around germinating bean (Phaseolus vulgaris L.) seeds, in the rhizosphere of bean, and in a model rhizosphere were studied. Strains of Rhizobium phaseoli that were resistant to streptomycin and thiram were used, and as few as 300 R. phaseoli cells per g of soil could be enumerated with a selective medium that was devised. A direct role was not evident for bacterial competitors, lytic bacteria, antibiotic-producing microorganisms, bacteriophages, and Bdellovibrio in the suppression of R. phaseoli around germinating seeds and in the rhizosphere. Protozoa increased in numbers in the soil upon planting of the seeds. The extent of colonization of soil by R. phaseoli was inversely related to the presence of large numbers of bacteria and protozoa. Colonization of R. phaseoli was improved upon suppression of protozoa with thiram and also when the soil was amended with other protozoan inhibitors and mannitol to simulate seed and root exudation. The data support the view that the decrease in numbers of R. phaseoli is caused by an increase in protozoan predation, the protozoa increasing in number because they prey on bacteria that proliferate by using seed and root exudates as nutrients.     

Enhancing Soybean Rhizosphere Colonization by Rhizobium japonicum

A study was conducted to seek means to increase the colonization of the rhizosphere of soybeans (Glycine max L. Merrill) by Rhizobium japonicum. For this purpose, a strain of R. japonicum that was resistant to benomyl, streptomycin, and erythromycin was used. The numbers of R. japonicum rose quickly in the first 2 days after soybean seeds were planted in soil and then rapidly fell. The decline was slower if the seeds were coated with benomyl. This fungicide reduced the numbers of bacteria and protozoa in the rhizosphere, but the effect became less or disappeared as the plants grew. In sterile soil inoculated with R. japonicum and a mixture of microorganisms, the numbers of R. japonicum were usually lower if protozoa were present than if they were absent. Nodulation and plant yield were increased by the addition of benomyl to soybean seeds sown in sterile soil inoculated with R. japonicum and a mixture of microorganisms. The addition of streptomycin and erythromycin to soil stimulated the growth of R. japonicum but inhibited other bacteria in the presence or absence of soybeans. The data indicate that colonization can be increased by the use of antimicrobial agents and R. japonicum strains resistant to those inhibitors.     

Effect of fungicides on survival of Rhizobium on seeds and the nodulation of bean (Phaseolus vulgaris L.)

The survival of Rhizobium leguminosarum biovar phaseoli on seeds of bean was tested, using the cultivar Carioca. The seeds were treated seven days before inoculation with Benlate, Vitavax, Banrot, Difolatan or Ridomil fungicides. The rhizobial strains used were: CIAT 899, CPAC 1135 and CIAT 652. Strain CIAT 899 showed greater survival on the seed with fungicide than the other strains. Two hours after the contact with fungicides strains CIAT 652 and CPAC 1135 had significantly lower numbers of rhizobia than the treatment without fungicide. The Benlate and Banrot fungicides had the greatest effect on survival of rhizobial strains. There was a drastic mortality of the two strains, CIAT 652 and CPAC 1135, on seeds treated with Benlate and Ridomil. Under field conditions, granular inoculation produced fewer nodules, but a similar total nodule weight as seed inoculation. Serological tests (ELISA) showed that seed treatment with Benlate in connection with seed inoculation reduced drastically the occurrence of inoculated strains in nodules, while the same fungicide treatment and inoculation applied in the seed furrow did not affect the survival of the inoculated strain.     

Population Changes and Persistence of Rhizobium phaseoli in Soil and Rhizospheres

The impact of legume cultivation on the establishment and persistence of an inoculant strain of Rhizobium phaseoli and its ability to compete with a resident population of R. phaseoli for nodule occupancy was examined utilizing strain-specific fluorescent antibodies. The soil (Hubbard loamy sand) was inoculated homogeneously with 5 × 10⁵ cells per g of soil and confined in plastic cylinders kept in field plots. Inoculated and uninoculated cylinders were either left fallow or planted to two seeds of legumes. Two hosts, navy bean (Phaseolus vulgaris L.) cv. Seafarer and snap bean cv. Picker, as well as a nonhost, soybean (Glycine max (L.) Merr.) cv. Wilkin, were used. Inoculant Viking 1 was highly stimulated in all three rhizospheres sampled at 6 (flowering), 10 (podfill), and 17 (decay) weeks and in the following spring, whereas counts in fallow soil decreased rapidly. Although the overwintering population remained highest in the vicinity of decaying host roots, Viking 1 persisted, even in fallow soil, to produce abundant nodulation of host plants the following spring. Viking 1 was an excellent competitor for nodulation sites on the roots of the hosts; it thoroughly outcompeted the resident population of R. phaseoli, occupying virtually 100% of the nodules under inoculated conditions in all experiments.     

Recognition of Leguminous Hosts by a Promiscuous Rhizobium Strain

The lima bean (Phaseolus lunatus L.) and the pole bean (Phaseolus vulgaris L.) are nodulated by rhizobia of two different cross-inoculation groups. Rhizobium sp. 127E15, a cowpea-type Rhizobium, can induce effective nodules on the lima bean and partially effective nodules on the pole bean. Rhizobium phaseoli 127K14 can induce effective nodules on the pole bean but does not reciprocally nodulate the lima bean. Root hairs of the lima bean when inoculated with Rhizobium sp. 127E15 showed tip curling and swelling and infection thread formation as observed by light microscopy and scanning electron microscopy. When lima bean root hairs were inoculated with R. phaseoli 127K14, no host-specific responses were observed. Pole bean root hairs that had been inoculated with R. phaseoli 127K14 or Rhizobium sp. 127E15 also showed tip curling and swelling and infection thread formation. Colonization of lima bean root hairs by Rhizobium sp. 127E15 and pole bean root hairs by R. phaseoli 127K14 or Rhizobium sp. 127E15 appeared to involve the elaboration of microfibrils. This study showed that when Rhizobium sp. 127E15 nodulates a host of a different cross-inoculation group, it elicits the same specific host responses as it does from a host of the same cross-inoculation group.     

Effect of pH on competition for nodule occupancy by type I and type II strains ofRhizobium leguminosarum bv.phaseoli

The effect of soil pH on the competitive abilities of twoRhizobium leuminosarum bv.phaseoli type I and one type II strains was examined in a nonsterile soil system.Phaseolus vulgaris seedlings, grown in unlimed (pH 5.2) or limed (pH 7.6) soil, were inoculated with a single-strain inoculum containing 1 × 10⁶ cells mL^–1 of one of the three test strains or with a mixed inoculum (1:1, type I vs. type II) containing the type II strain CIAT 899 plus one type I strain (TAL 182 or CIAT 895). At harvest, nodule occupants were determined. In a separate experiment, a mixed suspension (1:1, type I vs. type II) of CIAT 899 paired with either TAL 182 or CIAT 895 was used to inoculateP. vulgaris seedlings grown in sterile, limed or unlimed soil. The numbers of each strain in the rhizosphere were monitored for 10 days following inoculation. The majority of nodules (> 60%) formed on plants grown in acidic soil were occupied by CIAT 899, the type II strain. This pattern of nodule occupancy changed in limed soil. When CIAT 899 was paired with TAL 182, the type I strain formed 78% of the nodules. The number of nodules formed by CIAT 899 and CIAT 895 (56% and 44%, respectively) were not significantly different. The observed patterns of nodule occupancy were not related to the relative numbers or specific growth rates of competing strains in the host rhizosphere prior to nodulation. The results indicate that soil pH can influence which symbiotype ofR. leguminosarum bv.phaseoli will competitively nodulateP. vulgaris.     

Identification of Rhizobium phaseoli Strains That Are Tolerant or Sensitive to Soil Acidity

A study was conducted to determine whether the survival of Rhizobium phaseoli in acid soils could be predicted on the basis of the tolerance of the organism to acidity in culture. Of 16 strains tested, all grew in culture at pH 4.6, but only those that grew at pH 3.8 survived in soils having pH values of 4.1 to 4.6. Strains that tolerated the lowest pH values in culture were tolerant of the highest aluminum concentrations. In one acid soil, an acid-tolerant strain was unable to survive in numbers greater than 100/g, but the poor survival was not related to the level of extractable aluminum or manganese in the soil. Reproduction of an acid-tolerant strain of R. phaseoli was enhanced in the rhizosphere of Phaseolus vulgaris in both acid and limed soils, but stimulation of an acid-sensitive strain by the plant occurred only in the limed soil. These results indicate that cultural tests can be used to predict the ability of R. phaseoli to survive in acid soil.     

Nitrogen-Fixing Rhizobial Strains Isolated from Common Bean Seeds: Phylogeny,Physiology, and Genome Analysis

Rhizobial bacteria are commonly found in soil but also establish symbiotic relationships with legumes, inhabiting the root nodules, where they fix nitrogen. Endophytic rhizobia have also been reported in the roots and stems of legumes and other plants. We isolated several rhizobial strains from the nodules of noninoculated bean plants and looked for their provenance in the interiors of the seeds. Nine isolates were obtained, covering most known bean symbiont species, which belong to the Rhizobium and Sinorhizobium groups. The strains showed several large plasmids, except for a Sinorhizobium americanum isolate. Two strains, one Rhizobium phaseoli and one S. americanum strain, were thoroughly characterized. Optimal symbiotic performance was observed for both of these strains. The S. americanum strain showed biotin prototrophy when subcultured, as well as high pyruvate dehydrogenase (PDH) activity, both of which are key factors in maintaining optimal growth. The R. phaseoli strain was a biotin auxotroph, did not grow when subcultured, accumulated a large amount of poly-β-hydroxybutyrate, and exhibited low PDH activity. The physiology and genomes of these strains showed features that may have resulted from their lifestyle inside the seeds: stress sensitivity, a ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) complex, a homocitrate synthase (usually present only in free-living diazotrophs), a hydrogenase uptake cluster, and the presence of prophages. We propose that colonization by rhizobia and their presence in Phaseolus seeds may be part of a persistence mechanism that helps to retain and disperse rhizobial strains.     

Survival of Rhizobium in Acid Soils

A Rhizobium strain nodulating cowpeas did not decline in abundance after it was added to sterile soils at pH 6.9 and 4.4, and the numbers fell slowly in nonsterile soils at pH 5.5 and 4.1. A strain of R. phaseoli grew when added to sterile soils at pH 6.7 and 6.9; it maintained large, stable populations in soils of pH 4.4, 5.5, and 6.0, but the numbers fell markedly and then reached a stable population size in sterile soils at pH 4.3 and 4.4. The abundance of R. phaseoli added to nonsterile soils with pH values of 4.3 to 6.7 decreased similarly with time regardless of soil acidity, and the final numbers were less than in the comparable sterile soils. The minimum pH values for the growth of strains of R. meliloti in liquid media ranged from 5.3 to 5.9. Two R. meliloti strains, which differed in acid tolerance for growth in culture, did not differ in numbers or decline when added to sterile soils at pH 4.8, 5.2, and 6.3. The population size of these two strains was reduced after they were introduced into nonsterile soils at pH 4.8, 5.4, and 6.4, and the number of survivors was related to the soil pH. The R. meliloti strain that was more acid sensitive in culture declined more readily in sterile soil at pH 4.6 than did the less sensitive strain, and only the former strain was eliminated from nonsterile soil at pH 4.8; however, the less sensitive strain also survived better in limed soil. The cell density of the two R. meliloti strains was increased in pH 6.4 soil in the presence of growing alfalfa. The decline and elimination of the tolerant, but not the sensitive, strain was delayed in soil at pH 4.6 by roots of growing alfalfa.     

Initial and residual toxicity of soil-applied thiram on the vesicular-arbuscular mycorrhizal symbiosis

The effect of the non-systemic fungicide thiram on the vesicular-arbuscular mycorrhizal (VAM) symbiosis and on Leucaena leucocephala was evaluated in a greenhouse experiment. In the uninoculated soil treated with P at a level optimal for mycorrhizal activity, mycorrhizal colonization of roots was low, and did not change as the concentration of thiram in the soil increased with the from 0 to 1000 mg/kg. When this soil was inoculated VAM fungus Glomus aggregatum, with VAM colonization was enhanced significantly, but decreased increase in thiram concentration until it coincided with the level observed in the uninoculated soil. Similarly, symbiotic effectiveness was reduced, its expression delayed or completely eliminated with increase in the concentration of thiram. Amending soil to a P level sufficient for non-mycorrhizal host growth fully compensated for thiram-induced loss of VAM activity if the thiram levels did not exceed 125 mg/kg. In soil treated with 50 mg thiram/kg, the toxicity of the fungicide dissipated within 66 days of application. At higher concentrations, the toxicity of the chemical on the mycorrhizal symbiosis appeared to be enhanced.Contribution from the Hawaii Institute of Tropical Agriculture and Human Resources Journal Series No. 3716     

Studies on death of deteriorated barley seeds in soil

Seeds of barley cv. Golden Promise which had been deteriorated by storing at 20% moisture content and 30 °C and which all had viabilities >85% were treated with a slurry of calcium peroxide and with dusts of a mercurial and a non-mercurial fungicide. Calcium peroxide improved seedling emergence in wet soil conditions and deteriorated seeds benefited more than non-deteriorated seeds. The fungicide dusts had no effects. In controlled conditions at 20 °C, deteriorated seeds in wet soil died mainly within 2 days of sowing and they were more tolerant if they were placed in moist soil for 2 days before transfer to wet soil. Application of a mercurial fungicide and sterilizing the soil and the surface of seeds improved emergence but did not restore it to the level in moist soil. Fusarium culmorum, F. equiseti, Mucor spp. and Mortierella spp. dominated the fungus flora isolated from surface sterilised dead seeds in wet soil. When the fungi were inoculated to seeds, they increased the proportion which died in wet soil but only at high inoculum levels. It was concluded that they acted primarily as saprophytes depriving seeds of oxygen and secondarily as seed colonisers.     

The effect of Rhizobium inoculation and nitrogen fertiliser on nitrogen fixation and seed yield of dry beans (Phaseolus vulgaris)

The potential benefit to be derived from seed inoculation of Phaseolus vulgaris beans with effective strains of Rhizobium phaseoli, was investigated in field experiments over three years on a site low in soil nitrogen and lacking indigenous effective strains of R. phaseoli. Inoculation with R. phaseoli (strain RCR 3644) produced significant increases in nodulation, nitrogenase activity and plant growth in all experiments. In trials in 1978 and 1979, with cv. Seafarer, inoculation, in the absence of nitrogen fertiliser doubled seed yields. In 1978, the seed yields from inoculated beans without nitrogen fertiliser (1–6 t/ha) were not significantly different from those obtained with uninoculated beans receiving the optimum nitrogen fertiliser treatment of 120 kg N/ha (1–75 t/ha). In 1979, with lower rainfall favouring more efficient utilisation of nitrogen fertiliser, inoculation gave seed yields (1–88 t/ha) equivalent to those obtained with 60 kg N/ha (1–70 t/ha) but significantly less than with 120 kg N/ha (2–88 t/ha). More precise estimates from nitrogen response curves showed that inoculation supplied the fertiliser equivalent of 105 and 70 kg N/ha in 1978 and 1979 respectively. In both years, significant benefits were also obtained by the combination of inoculation and nitrogen fertiliser. In a separate experiment in 1979, with four R. phaseoli strains inoculated onto eight bean cultivars, three were highly effective nitrogen fixers on all cultivars. Two strains (RCR 3644 and NVRS 963A) each increased mean yields, in the absence of nitrogen fertiliser, from 1–39 t/ha uninoculated to c. 2–5 t/ha inoculated whilst strain RCR 3622 was outstanding with a mean yield of 3-0 t/ha. An analysis of the nitrogen content of seed showed that gains from nitrogen fixation were 37–57 kg N/ha/growing cycle for the combination RCR 3644 with cv. Seafarer. However, 106 kg N/ha/growing cycle was recorded for the combination RCR 3622 and cv. Aurora.     

Increased Effectiveness of Competitive Rhizobium Strains upon Inoculation of Cajanus cajan

A field study was conducted in lysimeters containing ¹⁵N-enriched soil to determine the effects of four competitive rhizobium strains upon yield parameters of pigeon peas (Cajanus cajan). The greatest differences observed were in seed yields; strain P132 effected the highest seed yield (121 ± 20 g per plant), and the control strain (indigenous rhizobia) effected the lowest yield (43.9 ± 8 g per plant). With the exception of seeds and pods, the dry matter weights were not different. Although there appeared to be no effect by inoculum strains on the fractional content of N derived from biological nitrogen fixation when the total plant biomass was considered, strains P132 and 401 partitioned more of the N derived from fixation into seeds and leaves than did the other strains. Because the seeds comprised the major portion of plant N, more total N and more N derived from biological nitrogen fixation (about half of total N) were found in plants inoculated with P132, whereas the smallest amount was found in the uninoculated controls. P132 was also the best competitor with respect to indigenous rhizobia and acounted for all of the nodules found on the plants in which it was inoculated.     

Influence of Captan on some microorganisms and microbial processes related to the nitrogen cycle

Captan was applied to laboratory-incubated agricultural soil and to bacterial cultures to determine its effects on total counts of soil microorganisms, nitrification, ammonification of urea and asymbiotic dinitrogen fixation. In Captan-treated soils, total count of fungi, bacteria and actinomycetes decreased significantly only at a relatively high fungicide concentration (1000 μg.g⁻¹). Fungi and actinomycetes were more affected than bacteria. While oxidation of ammonia in an enriched, actively nitrifying culture was almost totally inhibited by Captan, ammonification of urea in incubated soil was only partly depressed. The depressing effect of Captan was more pronounced in cultures of Micrococcus than in those of Proteus. Asymbiotic dinitrogen fixation in nutrient-ammended soil was promoted during the first week and depressed on prolonged exposure to the fungicide depending on its first concentration. In autoclaved Azotobacter-inoculated soil a similar but less pronounced effect was noticed. Fixation by Azotobacter caltures was insensitive to Captan. In contrast, growth ofRhizobium phaseoli, R. leguminosarum andR. japonicum in yeast-extract-mannitol medium was adversly affected by Captan, particularly at 200 μg.ml⁻¹. Nodulation of pea and mung bean (1 month old potted plants) grown from surface-sterilized inoculated seeds in aptan-treated soil was also significantly depressed. Both total number of nodules decreased with increasing concentration of the fungicide, but the inhibitory effect was more pronounced in the number of effective nodules.     

A mutation that blocks exopolysaccharide synthesis prevents nodulation of peas by Rhizobium leguminosarum but not of beans by R. phaseoli and is corrected by cloned DNA from Rhizobium or the phytopathogen Xanthomonas

Summary A Tn5-induced mutant strain of R. phaseoli which failed to synthesize exopolysaccharide (EPS) was isolated and was shown to induce normal nitrogen-fixing nodules on Phaseolus beans, the host of this Rhizobium species. The corresponding wild-type Rhizobium DNA was cloned in a wide host-range vector and by isolating Tn5 insertions in this cloned DNA, mutations in a gene termed pss (polysaccharide synthesis) were isolated. These were introduced by marker exchange into near-isogenic strains of R. leguminosarum and R. phaseoli which differed only in the identity of their symbiotic plasmids. Whereas the EPS-deficient mutant strain of R. phaseoli induced normal nitrogen-fixing nodules on Phaseolus beans, the same mutation prevented nodulation of peas by a strain of R. leguminosarum which normally nodulates this host. Further, it was found that DNA cloned from the plant pathogen Xanthomonas campestris pathover campestris could correct the defect in EPS synthesis in R. leguminosarum and R. phaseoli and also restored the ability to nodulate peas to the pss::Tn5 mutant strain of R. leguminosarum.     

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.     

Establishment of Bradyrhizobium japonicum for soybean by inoculation of a preceding wheat crop

On fields with no history of soybean (Glycine max (L.) Merr.) production, inoculation alone is often inadequate to provide for adequate nodulation the first time this crop is grown. The objective of this study was to determine if inoculation of spring wheat (Triticum aestivum L.) seed with Bradyrhizobium japonicum would lead to an increase of B. japonicum numbers in the soil, and improve nodulation of a subsequent soybean crop. In the greenhouse, wheat seed inoculation increased B. japonicum numbers from undetectable numbers to >9000 g^–1 of soil, whereas the numbers of introduced B. japonicum declined in unseeded pots. In the field, inoculation of wheat seed increased B. japonicum numbers in the soil from undetectable levels to >4000 g^–1 the following year. When soybean seed was inoculated, but grown in soil devoid of B. japonicum, nodules formed only near the point of seed placement. The heaviest nodulation, and widest distribution of nodules in the topsoil were found whenB. japonicum was established the year before by wheat seed inoculation, plus soybean seed inoculation. Wheat seed inoculation the year before growing soybean, combined with proper soybean seed inoculation, should provide for abundant nodulation the first time soybean is grown on a field.     

A seed treatment for the control of Pythium damping-off diseases and Peronospora parasitica on brassicas

In soil inoculated with Pythium ultimum or Pythium irregulare, seed treatment with either Apron 70 (=1 g metalaxyl and 1 g captan/kg seed) or thiram gave control of pre-emergence damping-off of Brussels sprout and cabbage seedlings. On cauliflower, Apron 70 was significantly more effective than thiram. No post-emergence damping-off occurred in either of these crops or in oil-seed rape following seed treatment with Apron 70 whilst post-emergence losses from untreated seed ranged from 10·2–19·4% and from thiram treated seed from 5·7-7·4%. Apron 70 gave complete control of Peronospora parasitica on cauliflower inoculated 10 days after sowing; thiram was ineffective. Following seed treatment with Apron 70, metalaxyl was detected in the cotyledons, true leaves and roots of cabbage seedlings up to 4 wk from sowing.     

Possible Involvement of Phage-Like Structures in Antagonism of Cowpea Rhizobia by Rhizobium trifolii

A reduction in the viability of cowpea rhizobia was observed when Rhizobium trifolii IARI and cowpea Rhizobium strain 3824 were inoculated together in soil. The reduction in number of cowpea rhizobia in soil was found to be associated with the reduction in number of nodules per plant and retardation in plant growth. An antimicrobial substance was isolated from R. trifolii which, on electron microscopic investigation, demonstrated the presence of several phage-like structures.     

Effectiveness of Rhizobium Strains Used in Inoculants after Their Introduction into Soil

Rhizobium strains used in inoculants for Trifolium spp., Medicago spp., Glycine max, and Lotus pedunculatus were isolated from nodules of these legumes grown in soils into which the rhizobia had been introduced 4 to 8 years before. Isolations were made from a total of 420 nodules. Nodule occupancy by the inoculant strains varied from 17.7% for a soybean strain to 100% in the case of L. pedunculatus whose specific rhizobia did not occur in the soils studied. In general, inoculant strains isolated from nodules did not differ in effectiveness from cultures of the same strains concurrently maintained in lyophilized form. The average effectiveness of all of the isolates (identified and unidentified) from a legume was 7.1 to 73.3% higher than that of the unidentified isolates alone, demonstrating the prolonged effect that a single-seed inoculation has on the rhizobial population in a soil which had not been planted with legumes before. Relatively weak recovery of a Rhizobium japonicum strain introduced into soil 4 years after soybean seed inoculated with a different strain had been planted in the same soil confirmed the advantage of a resident population over an introduced inoculant strain.