Rhizobia in tropical legumes—XIII. Biochemical basis of acid and alkali reactions

Three fast-growing rhizobia (Rhizobium meliloti isolated from Medicago saliva, R. trifolii from Trifolium subterraneum, and Rhizohium sp. from Leucaena leucocephala) and three slow-growing rhizobia R. japonicum from Glycine max, Rhizobium spp from Centrosema pubescens and Crotolaria anagyroides) were grown in defined media. The mean generation times of the fast-growing and slowgrowing strains were 3.8 h and 8.6 h respectively. Slow-growing organisms raised the initial pH of the defined medium while the fast-growing organisms lowered it. Rates of oxygen consumption tended to be higher in the slow-growing organisms.UMKL 19 (isolated from L. leucocephala) possessed all the normal reactions of fast-growing rhizobia but had a single sub-polar flagellum, similar to the three slow-growing strains studied.Certain combinations of amino acids and sugars (e.g. glutamine and galactose) induced an acidic reaction in the fast-growing organisms while the slow-growing ones changed the media to alkaline. Fast-growing organisms utilized more galactose for growth compared to slow-growing ones. Both types of organisms synthesized and released a wide range of amino acids into the medium.We suggest that pH changes produced by rhizobia growing on yeast-extract mannitol media are caused by the preferential utilization of sugars by fast-growing organisms and nitrogenous compounds by slow-growing ones.     

Rhizobia in tropical legumes—XIV. Ion uptake differences between fast- and slow-growing strains

We compared the uptake of nitrogen, potassium and phosphorus (as well as ¹⁴C-labelled mannitol, ³H-labelled glutamate, and ³²P-labelled phosphate) in three fast- and three slow-growing rhizobia. The fast-growing strains used were Rhizobium meliloti (isolated from Medicago sativa), R. trifolii (from Trifolium subterraneum), and Rhizobium spp from Leucaena leucocephala, while the slow-growing strains were R. japonicum (Glycine max), and two Rhizobium spp (from Centrosema pubescens and Crotolaria anagyroides). Slow-growing organisms preferentially utilized glutamate in the medium. Both fast- and slow-growing strains took up more NH⁺₄-N than NO^?₃-N on a per cell basis. In the presence of mannitol, fast-growing strains can cause either acid or alkaline reactions, an effect that is dependent only on the N-source (NH⁺₄ or NO^?₃). Uptake preferences of the fast-growing Leucaena isolate (UMKL 19) resembled those of the slow-growing rhizobia, further strengthening the argument that this organism (and others like it) may be intermediate between the normal fast- and slow-growing groups. Generally, the efficiency of uptake of N (either as NH⁺₄ or NO^?₃), P, and therefore K, was greater in the fast-growing organisms.     

Some factors affecting the survival of root-nodule bacteria on desiccation

The average number of survivors of fast-growing medic rhizobia (3 strains), fast-growing Rhizobium leguminosarum types (6 strains) and slow-growing species (9 strains) following desiccation of sandy soil inoculated with 10⁶ bacteria·g^?1 soil was 727, 795 and 15,682 bacteria·g^?1 soil, respectively. Survival in desiccated sandy soil was not influenced by the degree of extracellular polysaccharide production in strains of R. trifolii, nor was it influenced by growth of R. meliloti and slow-growing species in media of low water activity before desiccation in sandy soil.A progressive increase in numbers of fast-growing bacteria surviving desiccation was observed in sandy soil amended with increasing concentrations of powdered montmorillonite, but not with mont-morillonite added as a suspension to the soil. The clay had either a detrimental effect or no effect on the survival of the slow-growing rhizobia. Maltose, sucrose and polyvinylpyrrolidonc provided a greater degree of protection to both fast- and slow-growing rhizobia than was obtained with montmorillonite. The effect of polyethylene glycol 6000 was similar to the effect of montmorillonite, as the polymer only protected the fast-growing rhizobia and not the slow-growing species.     

Nodulation status of native woody legumes and phenotypic characteristics of associated rhizobia in soils of southern Ethiopia

The nodulation of provenances of Acacia seyal, Acacia tortilis and Faidherbia albida, and other indigenous multipurpose tree species were tested in 14 different soil samples collected from diverse agro-ecological zones in southern Ethiopia. Associated rhizobia were isolated from these and from excavated nodules of field standing mature trees, and phenotypically characterized. Indigenous rhizobia capable of eliciting nodules on at least one or more of the woody legume species tested were present in most of the soils. Tree species were markedly different in nodulation in the different site soils. Sesbania sesban and Acacia abyssinica showed higher nodulation ability across the different sites indicating widespread occurrence of compatible rhizobia in the soils. The nodulation patterns of the different provenances of Acacia spp. suggested the existence of intraspecific provenance variations in rhizobial affinity which can be exploited to improve N fixation through tree selection. Altogether, 241 isolates were recovered from the root nodules of trap host species and from excavated nodules. Isolates were differentiated by growth rate and colony morphology and there were very fast-, fast-, slow-, and very slow-growing rhizobia. The bulk of them (68.5%) were fast-growing acid-producing rhizobia while 25.3% were slow-growing alkali-producing types. Fast-growing alkali-producing (2.9%) and slow-growing acid-producing strains (3.3%) were isolated from trap host species and excavated nodules, respectively. All isolates fell into four colony types: watery translucent, white translucent, dull glistering and milky (curdled) type. The diversity of indigenous rhizobia in growth rate and colony morphology suggested that the collection probably includes several rhizobial genera.     

Symbiotic effectiveness of Bradyrhizobium japonicum in acid soils can be predicted from their sensitivity to acid soil stress factors in acidic agar media

In acid soil, low pH, reduced availability of nutrients, and toxicity of Al and Mn limit plant growth and the survival and effectiveness of rhizobia. The symbiosis between legumes and rhizobia is particularly sensitive to acid soil stress. A pot experiment evaluated whether Bradyrhizobium japonicum strain growth on acidic agar media would predict ability to colonize the rhizosphere and form effective nodules in acidic soils. Three Indonesian strains of B. japonicum with similar effectiveness at neutral pH in sand culture but with different tolerance of acid soil stress factors in agar media, and an acid-tolerant commercial strain (CB1809) of comparable effectiveness, were tested in three acid soils using the Al tolerant soybean (Glycine max cv PI 416937). At 7 days after inoculation all strains had achieved large rhizosphere populations, but by day 14 the rhizosphere population of the acid-sensitive strain had decreased, while the more acid-tolerant strains increased. The acid-tolerant strains had significantly greater nodulation and symbiotic effectiveness than plants inoculated with the acid-sensitive strain. Laboratory prescreening of B. japonicum for acid, Al and Mn tolerance in acid media successfully identified strains which were symbiotically competent in low pH soils.     

Rhizobial and bradyrhizobial symbionts of mesquite from the Sonoran Desert: salt tolerance, facultative halophily and nitrate respiration

Rhizobial symbionts were isolated from the surface (0-0.5 M) and phreatic (3.9-5.0 M) root environments of a mature mesquite woodland in the Sonoran Desert of Southern California, and from variable depths (0-12 m) of non-phreatic mesquite ecosystems in the Chihuahuan Desert of New Mexico. They were tested for their ability to tolerate high salinity, and respire NO₃⁻ as mechanisms of free-living survival. Sixteen of 25 isolates were grown in yeast-extract mannitol (YEM) broth at NaCl concentrations of 2 (basal concentration), 100, 300, 500 and 600 mM, and their specific growth rates, cell dry weight and lag times were determined. Twenty of the 25 isolates were also grown in YEM broth under anaerobic conditions with or without 10 mM KNO₃. Three categories of NaCl salinity responses were observed: (1) eight isolates showed decreased specific growth rates at NaCl concentrations of 100, 300 and 500 mM, but they nevertheless remained viable at 500 mM NaCl concentration; (2) the specific growth rate of six isolates increased significantly at 100 and 300 mM NaCl; and (3) specific growth rates of two isolates were significantly greater than the base-rate at all concentrations of NaCl. Five of 11 of the Bradyrhizobium isolates tested respired NO₃⁻, but showed no growth. Seven Rhizobium isolates, three from the deep (3.9-5 m) phreatic rhizobial community, and four from the surface community denitrified NO₃⁻ but only the isolates from the phreatic community displayed anaerobic growth. Long-term interactions between rhizobial and bradyrhizobial communities and the surface and phreatic root environments of the mature Sonoran Desert mesquite woodland appear to have selected for strains of NO₃⁻ respiring rhizobia, general salt tolerance of both rhizobial and bradyrhizobial symbionts, and strains of weak facultative halophilic bradyrhizobia. These survival characteristics of mesquite rhizobia may be important regarding mesquite's establishment and long-term productivity in marginal desert soils, and may provide novel types of rhizobia for food crops growing in harsh environments.     

Nodulation of Lotus pedunculatus in acid rooting solution by fast- and slow-growing rhizobia

The nodulation of Lotus pedunculatus and the multiplication of three Rhizobium loti (fast-growing, acid-producing) and two Bradyrhizobium (slow-growing, alkali-producing) strains was investigated in acidified rooting solution. R. loti strains multiplied at pH 4.5 but Bradyrhizobium strains failed to multiply. No difference in growth rate between R. loti and Bradyrhizobium strains was apparent in rooting solution at pH 6.7. Similar responses to pH were observed in yeast extract-mannitol broth except that Bradyrhizobium strains multiplied more slowly than R. loti at pH 6.7. All strains nodulated L. pedunculatus growing in acid (pH 4.5) rooting solution when presented as single cultures. Following inoculation with 1:1 mixtures of R. loti and Bradyrhizobium strains, R. loti formed 93% of nodules at pH 4.5 and significantly fewer nodules (66%) at pH 6.7. These results demonstrate a competitive advantage for acid-tolerant strains over acid-sensitive strains in nodulation of their lost legume at pH 4.5.     

Screening clover and Lotus rhizobia for tolerance of acidity and aluminium

Clover rhizobia (55 strains) were screened for tolerance of acidity and Al, using the technique of Keyser and Munns (1979). Assessment of visible turbidity after 14 days indicated three strains tolerant of pH 4.5 (although growth rate was reduced), 25 strains tolerant of 5μm Al and no strains tolerant of 50 μ m Al at pH 5.5.50 μmAl caused a decrease in the numbers of acid-tolerant strains at pH 4.5. Tolerance of acidity or Al was not associated with the pH or Al status of the soil from which a strain was isolated.Screening of eight strains of clover rhizobia and nine strains of Lotus rhizobia using turbidity assessment and viable counts indicated seven strains of clover rhizobia with different degrees of tolerance of 20 μm Al but none tolerant of 50 μm Al at pH 5.5. All Lotus rhizobia (both slow- and fast-growers) were tolerant of 20 and 50 μm Al at pH 5.5, with 50 μm Al causing a reduction in growth rate.Subculturing of strains in non-stressed and stressed media had no effect on the response to 50 μmAl at pH 5.5.     

Intrinsic antibiotic resistance and competition in fast- and slow-growing soybean rhizobia on a hybrid of Asian and US cultivars

Summary Six fast-growing soybean rhizobia (Rhizobium fredii) and thirteen slow-growing soybean rhizobia (Bradyrhizobium japonicum) were examined for resistance to 10 antibiotics. Axenic studies were carried out to determine the competitiveness of dual-strain inocula consisting of fast- and slow-growing rhizobia isolated from subtropical-tropical soils for nodule occupancy on a hybrid of Asian and US soybean cultivars. Nodule occupancy was determined by intrinsic resistance to erythromycin and neomycin. The results showed wide variability in resistance to 10 antibiotics for fast- and slow-growing rhizobia. The intrinsic antibiotic resistance of fast- and slow-growing rhizobia was extremely high against nalidixic acid (400 g ml^–1) and penicillin (200 g ml^–1). The competitive ability of inoculant strains for nodule occupancy varied for different combination sets and with the plant growing media. Our results show that fast-growing rhizobia nodulate a hybrid of Asian and US soybean cultivars. Fast-growing soybean rhizobia did not completely exclude nodulation by the slow-growing strains, which formed 0–79% nodules, depending on the strain used in the inoculum.     

Laboratory prescreening of Bradyrhizobium japonicum for low pH,Al and Mn tolerance can be used to predict their survival in acid soils

We examined whether strains of Bradyrhizobium japonicum selected for growth on acid media in vitro would also survive and grow better in acid soils. Four agar screening media for acid-tolerant rhizobia, which differed in the number of acid soil stresses imposed (pH, low calcium (Ca) and phosphorus (P), high aluminum (Al) and manganese (Mn)), were assessed for their effects on the survival of 14 Indonesian strains and two commercial strains of B. japonicum. Survival of B. japonicum in the agar media was compared with that in two acid soils. A repeat stab inoculation method which provided a declining range of inoculum cell number to 10³ cells per stab was used to assess the daily growth of the strains on the screening media at 5 pH levels (3.8, 4.2, 4.5, 5.0, and 6.8). The growth and survival of the 16 strains were then measured at days 1, 8, 18, and 28 after inoculation in two acid soils (pH 4.24 and 4.35) sterilized using γ-irradiation at 5.0 Mrad. Selectivity of the agar media improved as more acid stress factors were incorporated in the media. Those strains of Bradyrhizobium identified as acid, Al and Mn-tolerant in acidic agar media, also had better survival in the low pH soils. There was no relationship between acid or alkali production on agar media and acid tolerance on agar or in soil. There was no apparent relationship between symbiotic performance and acid tolerance, and one acid-tolerant strain was as effective as the commercial inoculant strain CB1809. The most acid-tolerant strain was also the most ineffective.     

Effect of strains of Rhizobium trifolii on the establishment of oversown white clover (Trifolium repens)

Strains of Rhizobium trifolii incorporated into commercial peat inoculants were compared for their effect on the establishment and growth of oversown white clover (Trifolium repens) on soils devoid of infective rhizobia.There were marked differences in numbers of seedlings establishing and clover dry matter production per hectare with the various strains. However, when adjusted to a constant number of established seedlings, dry matter production from all strains, apart from one strain at one site, were similar indicating that the strains did not appear to influence the growth of individual clover plants.The marked differences in establishment of clover inoculated with the various strains could not be accounted for by differences in the number of rhizobia in the peat inoculant.Selecting strains of rhizobia for ability to increase establishment is considered important where clover is oversown onto soils devoid of rhizobia.     

Mesquite rhizobia isolated from the Sonoran Desert: Physiology and effectiveness

A mesquite Rhizobium isolated from the Sonoran Desert (strain AZ-M1) and a commercial mesquite Rhizobium obtained from the Nitragin Company (strain 31A5) were chosen as superior strains from among many evaluated in a screening study of the efficiency of mesquite rhizobia. Both strains were fast-growing and acid-producing in denned media. The desert strain AZ-M1 had the shortest mean generation time of 3.5 h. Strain 31A5 grew better in broth amended with various sugars and amino acids, but generally produced less acid. The desert strain showed greater resistance to various antibiotics than did 31A5. In a greenhouse study N applied at high rates inhibited N₂ fixation when either strain was used as inoculant for mesquite seed. At low N rates, AZ-M1 fixed more N than 31A5. Total N, nodule weight, C₂H₂ reduction, mesquite shoot weight and root weight were all significantly increased when AZ-M1 was the applied inoculant. This study shows that the mesquite Rhizobium AZ-M 1 isolated from the Sonoran Desert is infective and effective on mesquite seedlings. Fast growth rate, acid production and high resistance to antibiotics in laboratory media may indicate the adaptation of this organism to its microbial niche in the Sonoran Desert.     

Inoculation with the native Rhizobium gallicum 8a3 improves osmotic stress tolerance in common bean drought-sensitive cultivar

Abstract

Symbiotic nitrogen fixation potential in common bean is considered to be low in comparison with other grain legumes. However, it may be possible to improve the nitrogen fixation potential of common bean using efficient rhizobia. In order to improve osmotic stress tolerance of a drought-sensitive common bean cultivar (COCOT) consumed in Tunisia, plants were inoculated either by the reference strain Rhizobium tropici CIAT 899 or by inoculation with rhizobia isolated from native soils Rhizobium gallicum 8a3. Fifteen days after sowing, osmotic stress was applied by means of 25 mM mannitol (low stress level) or by 75 mM mannitol (high stress level). Fifteen days after treatment plants were harvested and different physiological and biochemical parameters were analysed. Results showed no significant differences between the studied symbioses under control conditions. However after exposure to osmotic stress our results showed better tolerance of COCOT to osmotic stress when inoculated with the native R. gallicum 8a3. This can be partially explained by better water-use efficiency in both leaves and nodules, better relative water content in nodules and better efficiency in utilization of rhizobial symbiosis as compared with COCOT-CIAT 899 symbiosis. Hence, the present study suggested the better use of native soil isolated strains for the inoculation of common bean in order to improve its performance and nitrogen fixation potential under stressful conditions.     

Diversity in the nutritional requirements of strains of various Rhizobium species

An examination of 85 strains of bacteria from five species of rhizobia (Rhizobium sp., R. japonicum, R. lupini, R. meliloti and R. trifolii), using a new semi-quantitative assay procedure, disclosed wide diversity among the strains in their requirement for, and response to, vitamins, carbon sources, and nitrogen sources. Approximately half of the strains in the first four species grew as well without vitamins as they did when supplied with a vitamin mixture or with yeast extract, but the other strains showed considerable variation in their requirements. Some strains were inhibited by yeast extract, or showed best growth in basic media supplemented with only one vitamin. The strains within the species differed widely in their utilization of gluconate, mannitol and arabinose as C-sources; there was less diversity in their use of glutamine, histidine, NH₄⁺-N and NO₃^?-N as N-sources. The significance of these observations in the culture of rhizobia in the laboratory, in their ecological adaptation to particular environments, and in their ability to form an effective symbiosis with particular host legumes, is discussed.     

The potential for rhizobial inoculation to increase soybean grain yields on acid soils in Ethiopia

In Ethiopia, inoculation of soybean with rhizobial inoculants is not common practice, but could provide an option to increase grain yields in low nitrogen (N) acidic soils. In these acid soils, the selection of acid tolerant rhizobia is one strategy that may increase the performance of soybean. In this study, rhizobial strains isolated from Ethiopian soils were evaluated for their acid tolerance and symbiotic N fixation efficiency with soybean, in controlled environments. Following this, four isolated rhizobial strains were evaluated in six field experiments in major soybean growing areas of Ethiopia. Inoculation with the commercial strain or with one of two locally sourced isolates, that were developed as inoculants, improved soybean yield. The yield increase due to inoculation with the commercial strain was consistent and greater than other treatments, while the increase due to the two locally sourced strains was comparable to, or greater than, application of 46 kg N/ha in soils, where the resident rhizobial population was ≤1.4 × 10³ cfu/g soil. For soils with high background rhizobial populations, there was no response to inoculation. In one of the experimental sites (Bako), the percentage of N fixed (%Ndfa) was 55 for the commercial strain and 35 for the local strain, ES3. This study demonstrated that field validation is a necessary step in the selection of acid-tolerant strains of rhizobia to increase soybean production for Ethiopia.     

山西省快生型大豆根瘤菌资源调查和鉴定

从山西省主要大豆产区的不同土壤和大豆品种中分离得到的38个快生型大豆根瘸菌株的鉴定表明,这些分离物的IAR除了氨苄青霉素外,均较慢生型为低。38个菌株被分为4个血清型,其中2个为新发现的,命名为2077和2120型。细胞成分N%含量为2.01-3.78,C%含量为50.52-55.53%,N/C值<10。所检测的7个菌株都有1-2个大质粒,且每个均有112 Md的大质粒。分离株的共生效应和结瘤竞争由于大豆品种不同而有显著差异。本研究表明,我国大豆起源地之一的山西省,快生型大豆根瘸菌的分布广泛,分离频率较高,菌株类型也多。     

会泽铅锌尾矿区豆科植物根瘤菌耐铅锌性及其生理生化特征研究

研究铅锌尾矿区豆科植物根瘤菌对铅锌的响应,可为利用豆科植物改良尾矿区土壤及植被恢复提供理论依据。会泽铅锌尾矿土壤被铅锌严重污染,铅锌含量分别是全国土壤（A层）的110倍和54倍。通过现场采样、室内分离和耐性培养,对10株分离自会泽铅锌尾矿区豆科植物的根瘤菌的铅、锌单盐与铅锌双盐逆境进行了耐性研究。结果表明,该区域的豆科植物根瘤菌对铅、锌单盐逆境具有良好的耐性,但是对铅锌双盐的耐受明显减弱。对这10株根瘤菌的生理生化特征研究结果表明,会泽尾矿区根瘤菌的生理生化特性存在广泛差异,根瘤菌铅锌耐性越强,其阳性生理生化特征越多。一株与三叶草共生接瘤的HS3,一株与香豌豆共生接瘤的HX6和一株与猪屎豆共生接瘤的HZ8,它们不但表现出耐铅锌,而且生理代谢广泛,显示这3株根瘤菌在铅锌尾矿区的利用具有较好的潜能。     

Alternatives to peat as a carrier for rhizobia inoculants: Solid and liquid formulations

Many of the microbial inoculants all over the world are based on solid peat formulations. This has been mostly true for well developed legume inoculants based on selected rhizobial strains, due to peat bacterial protection properties. Six carriers (bagasse, cork compost, attapulgite, sepiolite, perlite and amorphous silica) were evaluated as alternatives to peat. Compost from the cork industry and perlite were superior to peat in maintaining survival of different rhizospheric bacteria. Other tested materials were discarded as potential carriers for soybean rhizobia. Also, different liquid culture media have been assayed employing mannitol or glycerol as C sources. Some media maintained more than 10⁹ cfu ml^?1 of Sinorhizobium (Ensifer) fredii SMH12 or Bradyrhizobium japonicum USDA110 after 3 months of storage. Rhizobial survival on pre-inoculated seeds with both solid and liquid formulations previously cured for 15 days led to a higher bacterial numbers in comparison with recently made inoculants. An additional curing time of solid inoculants up to 120 days had a beneficial effect on rhizobial survival on seeds. The performance of different formulations of two highly effective soybean rhizobia strains was assayed under field conditions. Soybean inoculated with cork compost, perlite and liquid formulations produced seed yields that were not significantly different to those produced by peat-based inoculants.     

Nodulation of Trifolium subterraneum by introduced rhizobia in competition with naturalized strains

The ability of 4 strains of Rhizobium trifolii to compete with naturalized strains in nodulating Trifolium subterruneum cv. Mt Barker and cv. Woogenellup was assessed at 5 sites in New South Wales. The populations of naturalized rhizobia at these sites ranged from 4 × 10⁶ rhizobia/g to one where no rhizobia were detected. The introduced strains were inoculated singly or as mixed strain inocula onto seed of the host at 2 × 10⁶ rhizobia/seed. There were marked differences in competitive ability between the strains but these differences were modified by the host cultivar and the site.At the R. trifolii-free site the inoculum strain formed 100% of the nodules in the 1st yr; by the second year serologically unrelated strains had invaded the plots and these formed almost all of the nodules in the 3rd yr. At the site where competition was greatest (4 × 10⁶ naturalized rhizobia/g), there were no differences in the competitive abilities of the strains in the first year but at all other sites WU95 was superior whether used as a single strain or in a mixed strain inoculum. In these sites also the proportion of nodules formed by the inoculum strains declined markedly by the 2nd yr.     

Diversity and symbiotic effectiveness of rhizobia isolated from field-grown soybean nodules in Paraguay

Soybean was introduced in Paraguay in the 1920s and commercial crops have been grown since the 1970s. Root nodulation occurs at the majority of the producing sites, although inoculation has been practiced in only 15-20% of the cropping areas. The diversity and symbiotic effectiveness of soybean rhizobia was studied using 78 isolates obtained from root nodules of field-grown plants at 16 sites located in the two main producing states. The rhizobial isolates were characterized in relation to several parameters in vitro (colony morphology, tolerance to high temperature and salinity, intrinsic resistance to antibiotics, synthesis of indole acetic acid, profiles of proteins and lipopolysaccharides) and in vivo (nodulation, plant growth and total N accumulated in shoots). Fifty-eight isolates had slow growth rates and alkaline reaction in medium containing mannitol as the carbon source, whereas 20 had fast growth rates and an acid reaction. Most isolates did not tolerate acidity (pH 4.5) or high temperature (40°C). Very few isolates shared similar protein and lipopolysaccharide profiles; therefore a high level of diversity was detected, with most of the isolates representing unique strains. Some of the isolates with an outstanding symbiotic performance were identified, and will now be tested under field conditions in a search for efficient and competitive strains for use in commercial inoculants in Paraguay.