Surviving and thriving in terms of symbiotic performance of antibiotic and phage-resistant mutants of Bradyrhizobium of soybean [Glycine max (L.) Merrill].

Rhizobial inoculation plays an important role in yielding enhancement of soybean, but it is frequently disturbed by competition with bacterial population present in the soil. Identification of potential indigenous rhizobia as competitive inoculants for efficient nodulation and N(2)-fixation of soybean was assessed under laboratory and field conditions. Two indigenous bradyrhizobial isolates (MPSR033 and MPSR220) and its derived different antibiotic (streptomycin and gentamicin) and phage (RT5 and RT6)-resistant mutant strains were used for competition study. Nodulation occupancy between parent and mutant strains was compared on soybean cultivar JS335 under exotic condition. Strain MPSR033 Sm(r) V(r) was found highly competitive for nodule occupancy in all treatment combinations. On the basis of laboratory experiments four indigenous strains (MPSR033, MPSR033 Sm(r), MPSR033 Sm(r) V(r), MPSR220) were selected for their symbiotic performance along with two exotic strains (USDA123 and USDA94) on two soybean cultivars under field conditions. A significant symbiotic interaction between Bradyrhizobium strains and soybean cultivar was observed. Strain MPSR033 Sm(r) V(r) was found superior among the rhizobial treatments in seed yield production with both cultivars. The 16S rRNA region sequence analysis of the indigenous strains showed close relationship with Bradyrhizobium yuanmingense strain. These findings widen out the usefulness of antibiotic-resistance marked phage-resistant bradyrhizobial strains in interactive mode for studying their symbiotic effectiveness with host plant, and open the way to study the mechanism of contact-dependent growth inhibition in rhizobia.

Genotypic characterization of phage-typed indigenous soybean bradyrhizobia and their host range symbiotic effectiveness.

Analysis of genetic diversity among indigenous rhizobia and its symbiotic effectiveness with soybean cultivar is important for development of knowledge about rhizobial ecology. In India, little is known about the genetic resources and diversity of rhizobia nodulating soybean. Indigenous bradyrhizobia isolated from root nodules of soybean plants, collected from traditional cultivating regions of two states (Madhya Pradesh and Uttar Pradesh) of India, were screened for bacteriophage sensitivity to identify successful broad host range symbiotic effectivity. Of 172 rhizobial isolates, 91 showed sensitivities to eight lytic phages and form ten groups on the basis of sensitivity patterns. The genetic diversity of 23 isolates belonging to different phage groups was assessed along with that of strains USDA123 and USDA94 by the restriction fragment length polymorphism (RFLP) analysis of 16S rDNA, intergenic spacer (IGS) (16S-23S rDNA), and DnaK regions. RFLP analysis of 16S rDNA formed 5 groups, whereas 19 and 9 groups were revealed by IGS and the DnaK genes, respectively. The IGS regions showed many amplified polymorphic bands. Nine isolates which revealed high RFLP polymorphism in the abovementioned regions (16S rRNA, IGS, DnaK) were used for 16S rRNA sequence analyses. The results indicate that taxonomically, all isolates were related to Rhizobium etli, Bradyrhizobium spp., and Bradyrhizobium yuanmingense. The doubling time of isolates varied from 9 h (MPSR155) to 16.2 h (MPSR068) in YM broth. Five isolates which did not show cross infectivity with isolated phage strains were studied for symbiotic efficiency. All isolates showed broad host range symbiotic effectiveness forming effective nodules on Vigna mungo, Vigna radiata, Vigna unguiculata, and Cajanus cajan. The present study provides information on genetic diversity and host range symbiosis of indigenous soybean rhizobia typed by different phages.

Morphology and general characteristics of bacteriophages infectious to Robinia pseudoacacia mesorhizobia.

Four phages infectious to Mesorhizobium strains were identified in soil samples taken from local Robinia pseudoacacia stands. Based on their polyhedral heads and short noncontractile tails, three of the phages, Mlo30, Mam12, and Mam20, were assigned to group C of Bradley's classification, the Podoviridae family, while phage Mlo1, with its elongated hexagonal head and a long flexible tail represented subgroup B2 bacteriophages, the Siphoviridae family. The phages were homogeneous in respect of their virulence, as they only lysed Mesorhizobium strains, but did not affect strains of Rhizobium or Bradyrhizobium. On the basis of one-step growth experiments, the average virus yield was calculated as approximately 10-25 phage particles for phages Mlo30, Mam12 and Mam20, and as many as 100-120 for phage Mlo1. The rate of phage adsorption to heat-treated cells showed differences in the nature of their receptors, which seemed to be thermal sensitive, thermal resistant, or a combination of the two. Only the receptor for phage Mlo30 was likely to be an LPS molecule, which was supported by a neutralization test. The smooth LPS with O-antigenic chains of the phage-sensitive M. loti strain completely reduced the bactericidal activity of virions at a concentration of 1 µg/ml. The molecular weights of phage DNAs estimated from restriction endonuclease cleavage patterns were in the range from approximately 39 kb for group C phages to approximately 80 kb for B2.

Isolation and symbiotic characteristics of two Tn5-derived phage-resistant Bradyrhizobium japonicum strains that nodulate soybean.

Using transponson Tn5 mutagenesis, two transconjugants of Bradyrhizobium japonicum with the properties of both phage resistance and ability to induce nodulation were isolated at the frequency of 0.02%. These transconjugants were tested for their symbiotic performance on soybean cv. JS335 under greenhouse and field conditions. Both phage-resistant mutants induced nodules (nod (+)), but the transconjugant B. japonicum E13 was ineffective in nitrogen fixation (fix (-)). Rhizobiophage presence in the inoculum of phage-resistant mutants did not influence the symbiotic effectiveness. The mixture of wild strain and phage in the inoculum caused reduced symbiotic performance under controlled conditions, while under a field environment phage (100 and 500 mul of approximately 10(8) particles ml(-1)) presence did not have any recognizable effect on increased nodule dry weight, nitrogenase activity, or foliar N(2) content. On the basis of restriction fragment length polymorphism analysis, phage-sensitive, less effective, homologous bradyrhizobia belonging to B. japonicum were detected in root nodules of both inoculated and uninoculated plants. Inoculation of a higher concentration of phage in the inoculum significantly reduced the symbiotic performance, while the lower concentration of phage did not show any effect on phage-susceptible, less effective, homologous bradyrhizobia or, thus, symbiotic efficiency under field conditions. The phage-resistant mutant B. japonicum A49 showed effective symbiosis as efficient as that of the wild strain. Inoculation of phage-resistant mutants with lytic phage may reduce the occupancy of phage-susceptible, ineffective/less effective/mediocre homologous bradyrhizobia strains under natural complex soil conditions.

Morphology and general characteristics of lytic phages infective on strains of Bradyrhizobium japonicum.

Biological characteristics of three isolated phages (SR1, SR2, and SR3) lytic against three Bradyrhizobium japonicum strains were studied. These phages had no cross-infectivity among the host strains. Phage morphology indicates that they belonged to Siphoviridae (long noncontractile tail; SR1 and SR2) and Podoviridae (short tail; SR3) classes of bacteriophages. Lytic cycle of phages studied under identical conditions showed a distinct adsorption rate (67.3-99.1%), latent period (150-300 min), rise period (60-150 min), and burst size (110-200 pfu/cell). Stability in liquids and inactivation by osmotic shock, thermal, and ultraviolet irradiation were also distinct in this heterogeneous phage group. Influence of soil factors such as temperature, soil moisture, soil pH, and degree of phage adsorption to the soil on phage survival was determined. Major percent of free infective phages were obtained after desorption of phages from soil. Overall, temperature appeared to be the most important parameter affecting rhizobiophage survival in the soil.

Differential symbiotic response of phage-typed strains of Bradyrhizobium japonicum with soybean cultivars.

In this study, native Bradyrhizobium strains were isolated from the host plant, Glycine max, harvested from fields in Madhya Pradesh, India, and were typed by lytic rhizobiophages. Eight indigenous (Soy2, ASR011, ASR031, ASR032, MSR091, ISR050, ISR076 and ISR078) and two exotic strains (USDA123 and CB1809), all of which evidenced a distinct reaction with six phages, were employed in this study. The symbiotic interaction of these strains was studied initially using soybean cultivar JS335 in a sand culture in a controlled environment, and the efficiency was assessed based on the nodule number, nodule dry weight, plant dry weight, nitrogenase activity, and total accumulation of N per plant. Symbiotic effectiveness was found to be highest with the native phage-sensitive isolate ASR011, whereas it was at a minimum with the phage-resistant isolates, ISR050 and ISR078. Additionally, the effectiveness of these strains was evaluated using six soybean cultivars belonging to different maturity groups; namely, Bragg, Lee, Pusa20, PK416, JS335 and NRC37. Analysis of variance data evidenced significant differences due to both symbionts, for the majority of the tested parameters. The CB1809, USDA123, and ASR011 strains evidenced relatively superior symbiotic effectiveness with soybean cultivars Bragg, Lee and JS335. Strain ISR078 evidenced no significant responses with any of the cultivars. The ASR031 strain performed moderately well with all tested cultivars. The symbiotic response of all the strains was quite poor with cultivar PK416. Our studies showed that a significant relationship existed between the phage sensitivity and symbiotic efficiency of the bacterial strains with the host-cultivars.