Inhibition of growth of Bradyrhizobium japonicum bacteroid by spermidine and spermine in yeast extract

Abstract

Bacteroids are defined as the symbiotic forms of Rhizobium or Bradyrhizobium cells in the root nodules of their legume host. The differentiation to bacteroids involves various physiological changes and may be associated with some genetical changes. Single-colony isolates from a nodule formed by a Bradyrhizobium strain often differed in their effectiveness and intrinsic antibiotic resistance (Weaver and Wright 1987; Ozawa unpublished data). Detailed analysis of the changes in the gene structure requires the isolation of individual bacteroids from a nodule. However it has been reported that the viability of bacteroids in culture is very low and only a small fraction of a bacteroid population could produce colonies on yeast extract-mannitol (YEM) agar (Sutton et al. 1977). Bergersen (1974) concluded that the reversion of bacteroids to the vegetative, growing form is very rare.     

Hydrogen metabolism and nitrogen fixation of soybean (Glycine max. L.) inoculated with different strains ofRhizobium spp.

Summary Hydrogenase activities and N₂-fixing capacities of soybean nodules (Glycine max. cv. Hodgson), inoculated with strains ofBradyrhizobium japonicum andRhizobium fredii from different geographical regions, were measured after 35 days of culture under controlled conditions. Of the strains tested, 47% induced nodules with bacteroids which recycled H₂. The data obtained suggest that H₂-recycling ability is not a major factor influencing early N₂-fixation which depends essentially on the precocity and intensity of the initial nodulation.     

Soybean preference for Bradyrhizobium japonicum for nodulation

For the increase of the occupation ratio of inoculum strain in the competition with indigenous rhizobia, the relationship between Rj-genotypes of soybean and the preference of Rj-cultivars for various types of rhizobia for nodulation was investigated by using the Rj ₂ Rj ₄-genotype of soybean isolated from the cross between the Rj ₂ Rj ₃-cultivar IAC-2 and Rj ₄-one Hill (Ishizuka et al. 1993: Soil Sci. Plant Nutr., 39, 79-86). Firstly, these Rj ₂ Rj ₄-genotypes were found to harbor the Rj ₃-gene. The Rj ₂Rj₃Rj₄-genotypes of soybean were considered to exhibit a more narrow microsymbiont range for nodulation than the Rj ₂ Rj ₃-and Rj₄-cultivars. Therefore, rhizobia were isolated from the nodules of various Rj-genotypes of soybeans grown in soils, and the preference of the Rj ₂ Rj ₃ Rj ₄-genotype for indigenous rhizobia was examined. The nodule occupancy of serotype 110 was significantly higher in the bacteroids of the nodules from the Rj ₂ Rj ₃ Rj ₄-rgenotypes than in those from the other genotypes, non Rj-, Rj ₂ Rj _3-, and Rj ₄-cultivars. These results demonstrated that the Rj ₂ Rj ³ Rj ⁴-genotype prefers more actively serogroup USDA110 to the others of rhizobia. Thus, Rj ₂ Rj ₃ Rj ₄-genotype is superior to non- Rj-, Rj ₂ Rj ₃-, and Rj ₄-genotypes for the formation of efficient nodules for nitrogen fixation.     

Searching for Myanmar indigenous Bradyrhizobium type C strains that best identify Rj4 genotypes in soybean

Bradyrhizobium species are symbiotic partners of soybean plants. However, some Bradyrhizobium bacteria do not form functional nodules on the roots of Rj₄ genotype soybean cultivars. Our objective was to identify the strains of Bradyrhizobium (i.e., type C strains) that are least competent to form nodules on the roots of this plant genotype. We checked (i) previously isolated type C strains of Myanmar Bradyrhizobium elkanii (MMY6-1, MMY6-2, and MMY6-5), (ii) previously isolated type C strains of Myanmarese Bradyrhizobium spp. (MMY3-5 and MMY3-7), and (iii) strain Is-34 of B. japonicum, for nodule formation when associated with Rj₄ and other Rj genotype soybeans. Strains in groups (i), (ii), and (iii) are known to be incompatible with Rj₄ soybean genotypes. MMY3-5 and MMY3-7 produced functional nodules when associated with Rj₄ and other Rj genotype soybean cultivars, except Hill (Rj₄) cultivar. The ratios of ineffective nodule numbers/total nodule numbers (I/T ratios) for MMY6-1, MMY6-2, MMY6-5, and Is-34 in association with Rj₄ soybean cultivars were > 0.5, demonstrating incompatibility between these bacterial strains and the Rj₄ genotype. Interestingly, the I/T ratios of MMY6-1 and MMY6-2 were higher than that of Is-34 in almost all Rj₄ soybean cultivars. Thus, the nodule-forming abilities of the B. elkanii strains MMY6-1 and MMY6-2 were strongly suppressed in Rj₄ soybean cultivars; these strains may therefore be useful to identify the Rj₄ genotype in soybean cultivars.     

Nitrogen fixation capacity and nodule occupancy by Bradyrhizobium japonicum and B. elkanii strains

 In a previous study soybean Bradyrhizobium strains, used in Brazilian studies and inoculants over the last 30 years, and strains adapted to the Brazilian Cerrados, a region frequently submitted to environmental and nutritional stresses, were analyzed for 32 morphological and physiological parameters in vivo and in vitro. A cluster analysis allowed the subdivision of these strains into species Bradyrhizobium japonicum, Bradyrhizobium elkanii and a mixed genotype. In this study, the bacteria were analyzed for nodulation, N₂ fixation capacity, nodule occupancy and the ability to increase yield. The goal was to find a relationship between the strain groups and the symbiotic performance. Two strains of Brazilian B. japonicum showed higher rates of N₂ fixation and nodule efficiency (mg of N mg^–1 of nodules) under axenic conditions. These strains also showed greater yield increases in field experiments when compared to B. elkanii strains. However, no differences were detected between B. japonicum and B. elkanii strains when comparing nodule occupancy capacity. The adapted strains belonging to the serogroup B. elkanii SEMIA 566, most clustered in a mixed genotype, were more competitive than the parental strain, and some showed a higher capacity of N₂ fixation. Some of the adapted strains, such as S-370 and S-372, have shown similar N₂ fixation rates and nodulation competitiveness to two Brazilian strains of B. japonicum. This similarity demonstrates the possibility of enhancing N₂ fixing ability, after local adaptation, even within B. elkanii species. Differences in the DNA profiles were also detected between the parental SEMIA 566 and the adapted strains by analyses with the ERIC and REP-PCR techniques. Consequently, genetic, morphological and physiological changes can be a result of adaptation of rhizobia to the soil. This variability can be used to select strains capable of increasing the contribution of N₂ fixation to soybean nutrition. Received: 28 May 1997     

Dual inoculation with strains of Bradyrhizobium japonicum and Azospirillum brasilense to improve growth and biological nitrogen fixation of soybean (Glycine max L.)

The effects of inoculation with Bradyrhizobium japonicum and Azospirillum brasilense strains on the growth of soybean were evaluated with regard to the estimation of N₂ fixation using the ¹⁵N isotope dilution technique. Inoculation, in general, increased the dry mass of soybean as well as nitrogen content. Dual inoculation with a mixture of B. japonicum and A. brasilense strains was superior over single inoculation with B. japonicum. Nitrogen fixed (Ndfa) varied according to inoculant and soil conditions. Percentages of nitrogen derived from air (% Ndfa) using a non-nodulating isoline were 72% and 76% for B. japonicum and B. japonicum plus A. brasilense, respectively, in non-sterile soil. A similar but higher trend was recorded in sterilized soil, in which the percentages of N₂ fixed were 81% and 86% for single and dual inoculation, respectively. The correlation coefficient between N₂ fixed and N uptake (r=0.94) and dry mass (r=0.89) was significant. Application of special bacterial inoculants in agricultural systems of Egypt seems to be a promising technology and could be used for improving soybean growth as well as soil fertility, thus minimizing environmental pollution. Received: 10 January 1996     

Serological properties and intrinsic antibiotic resistance of soybean bradyrhizobia isolated from Thailand

A group of Bradyrhizobium strains isolated from soybean plants in Thailand did not correspond to any known DNA homology groups of Bradyrhizobium japonicum and Bradyrhizobium elkanii reported by Hollis et al. (J. Gen. Microbiol., 123, 215–222, 1981). To clarify the phenotypic characteristics of the group, serological properties and intrinsic antibiotic resistance (IAR) profile of 94 Thai strains were compared with those of USDA and Japanese strains. Indirect ELISA tests for each Thai strain were performed agaiIl.st polyclonal antisera prepared against 15 USDA standard serotype strains of B. japonicum and B. elkanii. Among the 94 Thai strains tested, 36 which were previously identified as B. elkanii, with the exception of one strain, were strongly responsive to an antiserum prepared against USDA 31. The remaining 58 strains, with the exception of two strains, showed multiple cross reactions which were peculiar to the Thai strains. These serological reaction patterns did not correspond to any known serogroups labeled as B. japonicum and B. elkanii. In the IAR test, the taxonomically unknown Thai soybean bradyrhizobia exhibited a high level of resistance to neomycin (50 µg/mL), polymyxin (50 µg/mL), nalidixic acid (15 µg/mL), and kanamycin (15 µg/mL). Kanamycin could thus be useful in combination with neomycine and nalidixic acid for distinguishing between the unknown Thai strains and strains of B. japonicum and B. elkanii. Our results demonstrated that the unknown Thai strains were serologically and IAR-phenotypically remote from both B. japonicum and B. elkanii.     

Enrichment of natural 15N abundance in frankia-infected nodules

Abstract

The enrichment of ¹⁵N in the nodules of some N₂-fixing leguminous plants is an interesting finding (Shearer et al. 1982). The extent of ¹⁵N enrichment differed depending on the plant species (Shearer et al. 1982; Yoneyama 1987) and bacterial strains (Steele et al. 1983), and in soybeans it was apparently related to the nitrogen fixation efficiency (Shearer et al. 1984)     

Concomitant inhibition of nitrogen fixation and N-Phenyl-1-Naphthylamine uptake activity of bacteroids of a Bradyrhizohium japonicum strain by nitrate

Abstract

Effect of the treatment of soybean nodules with nitrate on the permeability of the outer membrane of bacteroids of Bradyrhizobium japonicurn strain 138NR was examined using the hydrophobic fluorescent probe N-phenyl-1-naphthylamine (NPN). The incorporation of NPN into the outer and inner membranes of the bacteroid cells isolated from nodules of soybean (Glycine max L. Merro cv. Tamahomare) was followed by the measurement of fluorescence at 30°C. The NPN-uptake activity of the bacteroids was expressed by two parameters, the initial rate of NPN-uptake, k, and the final fluorescence intensity, F _max. Acetylene reduction activity (ARA) of nodules, k, and F _max decreased exponentially during three weeks following the 20 d period after planting (DAP), resulting in high correlations between logarithmic transforms of k-values or F _max per bacteroid and those of ARA per bacteroid. Application of 20 mM KNO₃ to the nodulated roots at 24 DAP inhibited ARA of the roots by 85% of the control after 4 d of treatment. There were concomitant decreases in k and F _max by 75% and 65% of the control, respectively. These results suggest that treatment of soybean nodules with nitrate results in some changes in the permeability of the outer membrane of bacteroids with a concomitant decline of N₂-fixing activity of bacteroids.     

Effects of root exudates of Rj2Rj3- and Rj4-genotype soybeans on growth and chemotaxis of Bradyrhizobium japonicum

Almost all of the soybean cultivars (Glycine max L. Merr.) form nodules on their roots by infection with Bradyrhizobium japonicum. However, it has been observed that the soybean cultivars harboring nodulation conditioning genes, for instance Rj ₂, Rj ₃, and Rj ₄, do not form effective nodules with some strains of B. japonicum. Ishizuka et al. (1991b) classified the rhizobia isolated from field-grown soybeans into nodulation-types A, B, and C based on the compatibility with these Rj-soybean cultivars. Nodulation-type B is incompatible with Rj ₂ Rj ₃-cultivars and type C is incompatible with Rj ₄-cultivars. Nodulation-type A rhizobia are compatible with both Rj ₂ Rj ₃ - and Rj ₄-cultivars. Furthermore, the Rj-cultivars were found to prefer appropriate nodulation-type rhizobia for nodulation, that is, non-Rj-, Rj ₂ Rj ₃ -, and Rj ₄-cultivars prefer nodulation-types A, C, and B rhizobia, respectively (Ishizuka et al. 1991a). In the present study, in order to examine the mechanisms of preference of Rj-cultivars for rhizobial strains for nodulation, root exudates from Rj-cultivars were examined for their effects on the growth rate and chemotaxis of various types of B. japonicum strains.     

Soybean preference for Bradyrhizobium japonicum for modulation

Abstract

Caldwell and Vest (1968) planted soybeans (Glycine max L. Merr.) with various genotypes at Beltsville, USA, without inoculating them with Bradyrhizobium japonicum, and showed that soybeans preferred certain serotypes of rhizobial strains for nodulation. Recently, the authors have reported that soybeans carrying nodulation-conditioning genes preferred appropriate strains showing specific behavior for nodulation (Ishizuka et al. 1991). For instance, nodulation of soybean cv. Hardee which carries the nodulation-conditioning genes, Rj ₂ and Rj ₃, does not occur with B. japonicum USDA122, USDA33, Is-1, etc. Nodulation of cv. Hill which carries the Rj ₄ gene, does not occur with B. japonicum USDA61, Is-21, etc. while A62-2 which carries a recessive gene rj ₁, does not nodulate with almost any of the strains of B. japonicum. Therefore, the B. japonicum strains can be classified into three nodulation types based on the compatibility with these Rj-cultivars, that is, type A strains which effectively nodulated both Rj ₂ Rj ₃-cultivars and Rj ₄-ones, type B strains which did not nodulate the Rj ₂ Rj ₃-cultivars and type C strains which did not nodulate the Rj ₄-cultivars. When the nodulation types of the isolates from nodules of field-grown soybeans were examined, it was suggested that the Rj ₂ Rj ₃-cultivars and Rj ₄-cultivars preferred the type C and type B strains, respectively (Ishizuka et al. 1991).     

Soybean preference for Bradyrhizobium japonicum for nodulation

For examining the probability of increase in the occupation ratio of inoculated rhizobium in nodules, various Rj-soybean cultivars including the Rj ₂ Rj ₃ Rj ₄-lines of soybean were grown in a field of the Kyushu University Farm. Bradyrhizobium japonicum USDA110 that carries uptake hydrogenase (Hup⁺) was used as an inoculum. The relative efficiency of nitrogen fixation generally increased by the inoculation. However, there were no significant differences in the effects among the genotypes of the host plants. The occupation ratio of serogroup USDA110 in the nodules on the taproot of the inoculated plants was in the range of 77–100%, suggesting that the B. japonicum strain USDA110 infected taproots immediately after inoculation. The occupation ratios in the nodules on the lateral roots were 53–67, 40–86, 63–83, and 62–77% in inoculated plants of the non-Rj-, Rj ₂ Rj ₃-, Rj ₄-, and Rj ₂ Rj ₃ Rj ₄-genotypes, respectively, and they decreased in all the genotypes with the progression of growth. At the time of the first sampling, the occupation ratios on the lateral roots of these Rj ₂ Rj ₃ Rj ₄-genotypes showed values intermediate between those of IAC-2 (Rj ₂ Rj ₃) and Hill (Rj ₄) , which were the parent cultivars of the Rj ₂ Rj ₃ Rj ₄-lines, B340, B349, and C242. The reduction in the occupation ratio of the serogroup USDA110 for about 1 month after the first sampling was the lowest (0.13–0.16) in the Rj ₂ Rj ₃ Rj ₄-genotypes, excluding B349, followed by the non-Rj- and Rj ₂ Rj ₃-genotypes and highest (0.52–0.69) in the Rj ₄-genotypes, excluding Hill. Therefore, it was considered that the population of compatible rhizobia with host soybean plants increased in the rhizosphere with the progression of the development and growth. The results showed that with the expansion of the root area of host plants, the occupation ratio of type A rhizobia including the serogroup USDA110 was high. Therefore, the Rj ₂ Rj ₃ Rj ₄-genotypes were superior to other Rj-genotypes in terms of the inoculation effects of nodulation type A rhizobium, B. japonicum USDA110. However, the preference of the Rj ₂ Rj ₃ Rj ₄-genotype for serogroup USDA110 is not sufficient to rule out the competition with the other serogroups in this study. Therefore, the study should be centered on the isolation of more efficient (Hup⁺) and highly compatible rhizobial strains with the Rj ₂ Rj ₃ Rj ₄- genotypes.     

Seedling characteristics and retention of current photosynthates in leaves in relation to initial growth in pigeonpea (Cajanus cajan L. Millsp.) and cowpea (Vigna sinensis Endl.)

Improved varieties of pigeonpea (Cajanus cajan L. Millsp.) still retain some growth characteristics of perennial types of wild species such as slow seedling growth and rato on ability which are considered to be major yield-restricting characteristics when the growth period is shortened. Studies on the physiological background of slow initial growth may help improve the potential productivity of short-duration pigeonpea. It was found that the low seedling vigor of pigeonpea could be ascribed to the small seed size (Narayanan et al. 1981; Brakke and Gardner 1987), low rate of photosynthetic CO₂ fixation (Rawson and Constable 1981), and low rates of metabolic processes (Huber and Hanson 1992; Ito et al. 1996). However, few authors have related the slow growth to the retention of current photosynthates and leaf respiration during the night. In contrast, seedling growth of cowpea (Vigna sinensis Endl.) was reported to be vigorous (Brakke and Gardner 1987).

The objectives of this study were to characterize the initial growth of pigeonpea in comparison with cowpea. Two experiments were carried out, one dealing with growth analysis and the other with O2 uptake of leaf and retention of current photosynthates during the night.     

Evaluation of effective Myanmar Bradyrhizobium strains isolated from Myanmar soybean and effects of coinoculation with Streptomyces griseoflavus P4 for nitrogen fixation

In order to substitute the use of chemical fertilizers in legume production, there is a need for the production of rhizobial inoculants which are capable of being used as biofertilizers. To achieve this, an effective symbiotic nitrogen (N) fixation between legumes and root nodule bacteria will be essential. Evaluation of effective Myanmar Bradyrhizobium (Jordan 1982) strains isolated from Myanmar soybean (Glycine max L. Merr.) and effects of coinoculation with Streptomyces griseoflavus Krainsky 1914 P4 for N fixation were studied in pot experiments using sterilized vermiculite and Hoagland solution in the Phytotron (25°C and 70% relative humidity) with completely randomized design and three replicates. N fixation ability of soybean was evaluated by acetylene reduction activity (ARA) by gas chromatography. It was found that MAS23 showed a relatively high degree of stability and a high level of ARA per plant on both Yezin-3 and Yezin-6 soybean varieties. In the symbiotic relationship between Bradyrhizobium strains and P4 experiments, the treatments consisted of six Bradyrhizobium strains (MAS23, MAS33, MAS34, MAS43, MAS48 and USDA110) and Streptomyces griseoflavus P4 were evaluated with four Myanmar soybean varieties (Yezin-3, Yezin-6, Hinthada and Shan Sein). In the Yezin-3 soybean variety, the best treatment for ARA per plant was found in the dual inoculation of P4 and MAS34. In the Yezin-6 soybean variety, the highest nodule dry weight was found in dual inoculation of P4 with MAS34 but the highest ARA per plant was observed in the dual inoculation of P4 and MAS23. On the other hand, single inoculation of MAS43 and coinoculation of P4 with MAS48 were significantly higher in N fixation of Hinthada, and coinoculation of P4 with MAS33 was significant improvement of ARA per plant (P < 0.05) in Shan Sein soybean.     

Variability of nodulation and dinitrogen fixation capacity among soybean cultivars

 In order to identify soybean cultivars with higher biological N₂ fixation capacities, North American and Brazilian soybean [Glycine max (L.) Merrill] cultivars, belonging to maturity groups VI–VIII, were evaluated for nodulation parameters and N₂ fixation rates. The symbiotic performance of 152 cultivars was evaluated in pots containing 4 kg soil with an established population of the three Bradyrhizobium elkanii strains [29w (SEMIA 5019):SEMIA 566 : SEMIA 587, 22%:36%:34%] which are established in most Brazilian soils cultivated with soybean. Differences were verified among cultivars, with some accumulating up to twice as much nodule dry weight and N in tissues as others. The variability among cultivars was also confirmed when six of them were used in a field experiment, resulting in differences in nodulation, yield and total N accumulated in grains. The analysis of nodule occupancy in 12 cultivars grown either under sterile conditions and receiving a double inoculum and N-free nutrient solution, or in pots containing soil with an established population of bradyrhizobia, showed the preference of cultivars for specific strains. Received: 7 December 1998     

Field performance of three Bradyrhizobium japonicum strains with two soybean (Glycine max. L.) cultivars

Summary A field experiment was condutced in a clay loam soil to study the performance of three Bradyrhizobium japonicum strains; USDA 110, USDA 138 and TAL 379, in relation to their N₂-fixing potential and competitiveness on two soybean cultivars (Clark and Calland). Inoculation of soybean cultivars with these strains, either singly or in combination, induced significant increases in plant dry weight, N₂ fixation and seed yields. However, no significant differences were found between the rhizobial strains and/or their mixtures in N₂ fixation and increased seed yield for both cultivars. The two soybean cultivars gave similar responses to inoculation. No significant differences in seed yield were observed between Clark and Calland cultivars. The interaction between inoculant strain and soybean cultivar was not significant. The competition between strains for nodulation was assessed. Strain USDA 110 was the most competitive, followed by USDA 138. Strain TAL 379 was always less competitive on both cultivars. The incidence of double-strain occupancy of nodules varied from 8% to 40%.     

Diversity and geographical distribution of indigenous soybean-nodulating bradyrhizobia in Japan

Abstract

Genetic diversity and distribution of indigenous soybean-nodulating bradyrhizobia in Japan were investigated based on restriction fragment length polymorphism analysis of PCR product (PCR-RFLP) analysis of the 16S?23S rDNA internal transcribed spacer (ITS) region using Bradyrhizobium USDA strains as reference strains. Soil samples were collected from five field sites in Hokkaido, Fukushima, Kyoto, Miyazaki and Okinawa in Japan. A total of 300 isolates were derived from three Rj-genotype soybean cultivars, Akishirome (non-Rj), CNS (Rj ₂ Rj ₃) and Fukuyutaka (Rj ₄), and five field site combinations. The PCR products of the ITS region were digested with HaeIII, HhaI, MspI and XspI. Electrophoresed patterns were analyzed for phylogenetic relationship using Bradyrhizobium reference strains. Results revealed 22 RFLP patterns and 11 clusters. The RFLP patterns of the seven clusters were similar or identical to Bradyrhizobium japonicum USDA 6, 38, 110, 115, 123 and Bradyrhizobium elkanii USDA 76 and 94. Four minor clusters were independent from the clusters of the reference strains. The isolation ratio revealed the major clusters at each field site. These results suggested that major clusters of indigenous bradyrhizobia might be in the order Bj123, Bj38, Bj110, Bj6 and Be76 from the northern to southern regions in Japan.     

Field measurement of carbon dioxide evolution from soil by a flow-through chamber method using a portable photosynthesis meter

Extract

Since a rise in atmospheric carbon dioxide (CO₂) concentration is expected to lead to global warming, it is important to quantify the global carbon circulation. The CO₂ evolution rate from soil has usually been measured by one of three methods: 1) CO₂ absorption (Anderson 1982), where the evolved CO₂ is absorbed in an alkali solution and the content subsequently determined, 2) closed chamber (Rolston 1986) in which the CO₂ evolution rate is calculated from the increase of the CO₂ concentration in a closed chamber covering the soil surface, and 3) flow-through chamber (Rolston 1986) in which a fixed rate of ambient air is pumped through an open chamber and the difference in the. CO₂ concentration between the inlet and the outlet is measured. Although the CO₂ absorption method is very simple in terms of apparatus and procedure, the determined CO₂ evolution rate tends to be underestimated in cases where the evolved CO₂ is not fully absorbed in the alkali solution (Ewel et al. 1987; Sakamoto and Yoshida 1988), or overestimated in cases where the CO₂ concentration in the chamber is too low to stimulate microbial activity (Koizumi et al. 1991; Nakadai et al. 1993), In the closed chamber method, when the gas concentration in the chamber is higher than that of the ambient air, gas diffusion from the soil to the atmosphere is restricted (Denmead 1978). At this point, the flow-through chamber method seems to be most suitable for measuring the CO₂ evolution rate, because the rate is determined under nearly natural conditions. However, this method has a disadvantage in that the apparatus is composed of an infra-red CO₂ analyzer, air pumps, mass flow meters, a recorder, and other items, which are too large, heavy, and complex to use in the field (Freijer and Bouten 1991). Hence, in spite of the above limitations, most of the studies on CO₂ evolution in situ have been carried out using the CO₂ absorption method (Kowalenko et al. 1978; Seto et al. 1978a, b; Ewel et al 1981, 1987; Gupta and Singh 1981; Reinke et al. 1981; Edwards and Ros-Todd 1983; Grahammer et al. 1991) or the closed chamber method (Naganawa et al. 1989; Mariko et al. 1994). The flow-through chamber method has been used only at sites where electric power supply and other types of equipment were available (Mathes and Schriefer 1985; Ewel et al. 1987; Nakadai et al. 1993). In the present report a flow-through chamber method using a portable CO₂ analyzer system was examined, for the determination of CO₂ evolution from soil without an electric power supply or other special equipment.     

Survival and symbiotic properties of Bradyrhizobium japonicum in the presence of thiram: isolation of fungicide resistant strains

The fungicide thiram, widely used as a chemical seed protectant, induces a strong inhibition of primary nodulation in the crown zone of soybean roots. The present work reports on the isolation of Bradyrhizobium japonicum strains resistant to thiram, some of which (T3B, A86 and A2) maintained their capacity for nodulation and were still efficient symbionts, but some (A1, C1 and C6) lost the ability to stimulate nodulation. Characterization tests such as growth at different pH, denitrifying ability, salt tolerance, production of siderophores and phosphate solubilization were performed on the resistant strains. Inoculants produced from these strains could be appropriate for use with thiram-treated seeds, without causing a loss of bacteria viability. Received: 16 September 1996