Effect of dinitroaniline herbicides on rhizobia, nodulation and N2(C2H2) fixation of four groundnut cultivars

Field, greenhouse and laboratory investigations were conducted to determine the effect of four dinitroaniline herbicides on rhizobia, nodulation and nitrogen fixation of four groundnut cultivars. Benefin, dinitramine and nitralin used at recommended levels decreased nodule dry weight, nitrogenase activity and total nitrogen of groundnut tops and pod yield in three cultivars Kadiri 71-1, Kadiri-2, ICGS-11 and not for a fourth cultivar, Kadiri-3 of groundnut (Arachis hypogaea L.), but fluchloralin used at the recommended level increased the nodulation rate, nitrogenase activity and total nitrogen of groundnut tops and pod yield compared to untreated plants. Studies were conducted in vitro to determine the relative toxicity of the herbicides on four Rhizobium strains isolated from the nodules of four cultivars of groundnut. It was found that various strains of rhizobia differ in their sensitivity to different rates of the herbicides tested. Carbon dioxide exchange rate (CER) of all the cultivars which received herbicide treatment was measured at different time intervals to determine the relationship between photosynthesis and inhibition of nodulation. The lack of adverse effect on the CER of four cultivars when treated at recommended concentrations indicated that nitrogen fixation was affected in cultivars Kadiri 71-1, Kadiri-2 and ICGS-11 due to inhibition of nodulation.     

Nodulation of lucerne (Medicago sativa L.) in an acid soil: Effects of inoculum size and lime-pelleting

The effects of inoculum level and lime-pelleting were studied in an acid soil with respect to the nodulation and growth of lucerne (Medicago sativa cv Resis) and the population dynamics of Rhizobium meliloti. In small root-boxes (rhizotrons), the in-situ survival of inoculated rhizobia was studied in the micro-environment around the seed for a period of 12 days after sowing. During the initial 24 hours, a strong increase in rhizobial numbers was measured, concomitantly with the development of roots. As a result of lime-pelleting, rhizobial numbers were higher only at 3 days after sowing (P<0.05). Later, this difference diminished steadily. Addition of lime did not increase the adhesion of the rhizobia to the seedling tap root. Plant responses to inoculation were studied in pots. To obtain optimal nodulation, the soil had to be neutralized around the seed with lime and at least 10⁵ cells of R. meliloti were required. With more than 10⁵ rhizobia per seed, lime-pelleting increased the number of crown-nodulated seedlings from 24% to 77%. Higher numbers of rhizobia could not compensate the effect of lime. A strong correlation was found between crown nodulation, nitrogen content and dry weight of the shoots.     

A dry granular inoculant of Rhizobium for soil application

A dry granular inoculant of Rhizobium was prepared from sodium alginate and peralite. High numbers of two groundnut (Arachis hypogaea) Rhizobium strains, NC 92 and TAL 1000 used to prepare inoculants survived in dry granules beyond 180 days. The viable counts were 9.72 and 9.91 log₁₀ rhizobia g^-1 of dry granules for NC 92 and TAL 1000, respectively compared to 8.0 log₁₀ rhizobia g^-1 of peat inoculant for NC 92 at the end of six months storage. The granular inoculant was free from contaminants. In a pot culture experiment the granular inoculant applied to the soil gave similar results when seeds were dressed with a peat inoculant; nodulation and growth of groundnut were similar. The major advantage of this inoculant is that, it can be stored in a dry state without losing much viability.     

Inoculation of pea by application of Rhizobium in the planting furrow

Summary Selected streptomycin resistant strains ofRhizobium leguminosarum suspended in nutrient broth were added to the planting furrow immediately before the sowing of pea. The nodule occupancy by a strain isolated from Risø soil (Risø la) was increased from 74 to 90%, when the inoculum rate was increased from 3.7×10⁶ to 3.7×10⁸ cells per cm row. The experimental soil contained 10³ to 10⁴ cells ofR. leguminosarum per gram. An almost inefficient strain isolated from Risø soil (SV10) was less competitive with respect to nodulation on two pea cultivars than an efficient Risø strain (SV15) and an efficient non-Risø strain (R1045). The nodule occupancy by the introduced strains varied between pea cultivars.Irrespective of the generally high nodulation by the efficient strains introduced to the soil, the pea seed yield, compared to pea nodulated by the indigenous population, was not significantly increased. Neither were two commercial inoculants, applied in rates corresponding to 3 times the recommended rate, able to increase the yield. This suggests that the indigenous populations ofR. leguminosarum were sufficient in number and nitrogen fixing capacity to ensure an optimal pea crop. However, some inoculation treatments slightly increased the seed N concentration and total N accumulation, indicating that it may be possible to select or develop bacterial strains that may increase the yield.     

Competition Among Rhizobium spp. for Nodulation of Leucaena leucocephala in Two Tropical Soils

The successful nodulation of legumes by a Rhizobium strain is determined by the competitive ability of that strain against the mixture of other native and inoculant rhizobia. Competition among six Leucaena rhizobial strains in single and multistrain inoculants were studied. Field inoculation trials were conducted in an oxisol and a mollisol soil, both of which contained indigenous Leucaena-nodulating rhizobia. Strain-specific fluorescent antibodies were used for the identification of the strains in Leucaena nodules. Mixtures of three recommended inoculum strains for Leucaena spp. (TAL82, TAL582, and TAL1145) were used in peat-based inocula either alone or with one of the three other strains isolated from the sites, B213, B214, and B215. Each of these latter three strains was also used as single-strain inocula to study their competition with the native rhizobia in the two soil systems. In the oxisol soil, strains B213 and B215, when used as single-strain inocula, outcompeted the native rhizobia and formed 92 and 62% of the nodules, respectively. Strain B214 was the least competitive in oxisol soil, where it formed 30% of the nodules, and the best in mollisol soil, where it formed 70% of the nodules. The most successful competitor for nodulation in multistrain inocula was strain TAL1145, which outcompeted native and other inoculum Leucaena rhizobia in both soils. None of the strains in single or multistrain inoculants was capable of completely overcoming the resident rhizobia, which formed 4 to 70% of the total nodules in oxisol soil and 12 to 72% in mollisol soil. No strong relationship was detected between the size of the rhizosphere population of a strain and its successful occupation of nodules.     

Interactive effects of synthetic nitrogen fertilizer and composted manure on ammonia volatilization from soils

The mutualism between legumes and nitrogen-fixing soil bacteria (rhizobia) is a key feature of many ecological and agricultural systems, yet little is known about how this relationship affects aboveground interactions between plants and herbivores. We investigated the effects of the rhizobia mutualism on the abundance of a specialized legume herbivore on soybean plants. In a field experiment, soybean aphid (Aphis glycines) abundances were measured on plants (Glycine max) that were either (1) treated with a commercial rhizobial inoculant, (2) associating solely with naturally occurring rhizobia, or (3) given nitrogen fertilizer. Plants associating with naturally occurring rhizobia strains exhibited lower aphid population densities compared to those inoculated with a commercial rhizobial preparation or given nitrogen fertilizer. Genetic analyses of rhizobia isolates cultured from field plants revealed that the commercial rhizobia strains were phylogenetically distinct from naturally occurring strains. Plant size, leaf nitrogen concentration, and nodulation density were similar among rhizobia-associated treatments and did not explain the observed differences in aphid abundance. Our results demonstrate that plant–rhizobia interactions influence plant resistance to insect herbivores and that some rhizobia strains confer greater resistance to their mutualist partners than do others.     

Agro-industrial waste materials and wastewater sludge for rhizobial inoculant production: a review

Inoculating legumes with commercial rhizobial inoculants is a common agriculture practice. Generally, inoculants are sold in liquid or in solid forms (mixed with carrier). The production of inoculants involves a step in which a high number of cells are produced, followed by the product formulation. This process is largely governed by the cost related to the medium used for rhizobial growth and by the availability of a carrier source (peat) for production of solid inoculant. Some industrial and agricultural by-products (e.g. cheese whey, malt sprouts) contain growth factors such as nitrogen and carbon, which can support growth of rhizobia. Other agro-industrial wastes (e.g. plant compost, filtermud, fly-ash) can be used as a carrier for rhizobial inoculant. More recently, wastewater sludge, a worldwide recyclable waste, has shown good potential for inoculant production as a growth medium and as a carrier (dehydrated sludge). Sludge usually contains nutrient elements at concentrations sufficient to sustain rhizobial growth and heavy metals are usually below the recommended level. In some cases, growth conditions can be optimized by a sludge pre-treatment or by the addition of nutrients. Inoculants produced in wastewater sludge are efficient for nodulation and nitrogen fixation with legumes as compared to standard inoculants. This new approach described in this review offers a safe environmental alternative for both waste treatment/disposal and inoculant production.     

Survival of several <Emphasis Type="Italic">Rhizobium/Bradyrhizobium</Emphasis> strains on different inoculant formulations and inoculated seeds

The effect of a variety factors on the survival of several rhizobia strains on inoculants and inoculated seeds has been evaluated. Since the rhizobia strains showed different cell-density-evolution patterns on peat-based inoculants and on inoculated seeds, several inoculant formulations with highly effective Rhizobium/Bradyrhizobium strains (for Lupinus, Hedysarum, Phaseolus and Glycine max.) were monitored under the following storage conditions: (a) the inoculants were kept refrigerated (at 4 °C), or (b) at room temperature (25 °C). The effect of water content (30–50%, w/w) in the inoculants as well as that of several seed-coating adhesives were also investigated. Alternative carriers including perlite and vermiculite were tested. For all of the strains, survival on sterile peat-based inoculants was higher than on the corresponding unsterile peat formulation; for the latter, refrigerated storage conditions are recommended to ensure high bacterial densities. The water content of the inoculants had a differential effect on strain survival depending on the sterility of the peat, such that a high water content was more detrimental when unsterilized peat was employed. The best adherent for rhizobia survival was a gum arabic/water solution. Perlite was as effective as peat in maintaining a high population of rhizobia, at least for 6 months of storage. Electronic Publication     

Chemotaxis,induced gene expression and competitiveness in the rhizosphere

Rhizobia are soil bacteria which symbiotically infect legume roots and generate nodules in which they fix atmospheric nitrogen for the plant in exchange for photosynthetically fixed carbon. A crucial aspect of signal exchange between these symbionts is the secretion of phenolic compounds by the host root which induce nodulation gene expression in the bacteria. Stimulation of nod gene expression by host phenolics is required for nodule formation, is biochemically specific at 10^-6 M, and is mediated by nodD. We and others have shown that rhizobia display chemotaxis to 10^-9 M of the same phenolic compounds. Chemotaxis to inducer phenolics is selectively reduced or abolished by mutations in certain nod genes governing nodulation efficiency or host specificity. Conversely, mutations in rhizobia that affect general motility or chemotaxis have substantial effects on nodulation efficiency and competitiveness. These findings suggest that microbes entering the rhizosphere environment may utilize minor, non-nutrient components in root exudates as signals to guide their movement towards the root surface and elicit changes in gene expression appropriate to this environment.     

Number of Rhizobia and delayed inoculation influence nodulation of clovers

The relationship between numbers of rhizobia and nodulation response of legumes is of considerable practical importance. Experiments were done under controlled conditions to determine the influence of numbers of Rhizobium leguminosarum biovar. trifolii on nodulation of arrowleaf clover (Trifolium vesiculosum Savi.) and crimson clover (T. incarnatum L.). Numbers of rhizobia in excess of 1000 per seed did not substantially increase earliness of nodulation or total number of nodules formed on the taproot. Nodules, however, were formed nearer the top of the taproot as numbers of rhizobia increased to 100,000 per seed. Delayed inoculation experiments indicated that nodulation sites for these clovers only remained susceptible to infection for less than 1 day. Delaying inoculation for 4 days resulted in only a 1 to 2 day delay in nodulation for arrowleaf and crimson clovers respectively and no delay for subterranean clover (T. subterraneum L.). Apparently, larger seedlings nodulated faster.     

Recent development and new insight of diversification and symbiosis specificity of legume rhizobia: mechanism and application

Currently, symbiotic rhizobia (sl., rhizobium) refer to the soil bacteria in α- and β-Proteobacteria that can induce root and/or stem nodules on some legumes and a few of nonlegumes. In the nodules, rhizobia convert the inert dinitrogen gas (N₂) into ammonia (NH₃) and supply them as nitrogen nutrient to the host plant. In general, this symbiotic association presents specificity between rhizobial and leguminous species, and most of the rhizobia use lipochitooligosaccharides, so called Nod factor (NF), for cooperating with their host plant to initiate the formation of nodule primordium and to inhibit the plant immunity. Besides NF, effectors secreted by type III secretion system (T3SS), exopolysaccharides and many microbe-associated molecular patterns in the rhizobia also play important roles in nodulation and immunity response between rhizobia and legumes. However, the promiscuous hosts like Glycine max and Sophora flavescens can nodulate with various rhizobial species harbouring diverse symbiosis genes in different soils, meaning that the nodulation specificity/efficiency might be mainly determined by the host plants and regulated by the soil conditions in a certain cases. Based on previous studies on rhizobial application, we propose a ‘1+n−N’ model to promote the function of symbiotic nitrogen fixation (SNF) in agricultural practice, where ‘1’ refers to appreciate rhizobium; ‘+n’ means the addition of multiple trace elements and PGPR bacteria; and ‘−N’ implies the reduction of chemical nitrogen fertilizer. Finally, open questions in the SNF field are raised to future think deeply and researches.     

Nitrate effects on the nodulation of legumes inoculated with nitrate-reductase-deficient mutants of Rhizobium

The effect of nitrate on the symbiotic properties of nitrate-reductase-deficient mutants of a strain of cowpea rhizobia (32H1), and of a strain of Rhizobium trifolii (TA1), were examined; the host species were Macroptilium atropurpureum (DC.) Urb. and Trifolium subterraneum L. Nitrate retarded initial nodulation by the mutant strains to an extent similar to that found with the parent strains. It is therefore unlikely that nitrite produced from nitrate by the rhizobia, plays a significant role in the inhibition of nodulation by nitrate. Nitrite is an inhibitor of nitrogenase, and its possible production in the nodule tissue by the action of nitrate reductase could be responsible for the observed inhibition of nitrogen fixation when nodulated plants are exposed to nitrate. However, the results of this investigation show that nitrogen fixation by the plants nodulated by parent or mutant strains was depressed by similar amounts in the presence of nitrate. No nitrite was detected in the nodules. Nodule growth, and to a lesser extent, the nitrogenase specific activity of the nodules (mol C₂H₄g^–1 nodule fr. wt. h^–1), were both affected by the added nitrate.     

The effectiveness of some Indonesian strains of Rhizobium on four tropical legumes

Nodules were collected from 14 legume species from the Indonesian Islands of South Sulawesi, Java and Sumatra. Their rhizobia were isolated and growth characteristics, nodulation ability and nitrogen fixing effectiveness were assessed against recommended commercially available Australian strains. The test legumes wereMacroptilium atropurpureum Urb. cv. Siratro,Vigna unguiculata (L.) Walp. cv Eureka,Centrosema pubescens Benth cv. Belalto andDesmodium heterocarpon (L) DC. A significant portion of the native rhizobial isolates were of the fast growing type. Dry matter and total nitrogen production forM. atropurpureum andV. unguiculata was highest when inoculated with native strains while the commerical strains produced superior dry matter production forC. pubescens andD. heterocarpon. However the total nitrogen production of native and commercial strains was not significantly different for the latter two legumes. The study indicated that a potential exists for developing inocula from local Rhizobium strains.     

Nod factors of Rhizobium are a key to the legume door

Symbiotic interactions between rhizobia and legumes are largely controlled by reciprocal signal exchange. Legume roots excrete flavonoids which induce rhizobial nodulation genes to synthesize and excrete lopo-oligosaccharide Nod factors. In turn, Nod factors provoke deformation of the root hairs and nodule primordium formation. Normally, rhizobia enter roots through infection threads in markedly curled root hairs. If Nod factors are responsible for symbiosis-specific root hair deformation, they could also be the signal for entry of rhizobia into legume roots. We tested this hypothesis by adding, at inoculation, NodNGR-factors to signal-production-deficient mutants of the broad-host-range Rhizobium sp. NGR234 and Bradyrhizobium japorticum strain USDA110. Between 10 ⁻⁷ M and 10⁻⁶ M NodNGR factors permitted these NodABC mutants to penetrate, nodulate and fix nitrogen on Vigna unguiculata and Glycine max, respectively. NodNGR factors also allowed Rhizobium fredii strain USDA257 to enter and fix nitrogen on Calopogonium caeruleum, a non-host. Detailed cytological investigations of V. unguiculata showed that the NodABC mutant UGR AnodABC, in the presence of NodNGR factors, entered roots in the same way as the wild-type bacterium. Since infection threads were also present in the resulting nodules, we conclude that Nod factors are the signals that permit rhizobia to penetrate legume roots via infection threads.     

Plant Growth-Promoting Rhizobacteria Allow Reduced Application Rates of Chemical Fertilizers

The search for microorganisms that improve soil fertility and enhance plant nutrition has continued to attract attention due to the increasing cost of fertilizers and some of their negative environmental impacts. The objectives of this greenhouse study with tomato were to determine (1) if reduced rates of inorganic fertilizer coupled with microbial inoculants will produce plant growth, yield, and nutrient uptake levels equivalent to those with full rates of the fertilizer and (2) the minimum level to which fertilizer could be reduced when inoculants were used. The microbial inoculants used in the study were a mixture of plant growth-promoting rhizobacteria (PGPR) strains Bacillus amyloliquefaciens IN937a and Bacillus pumilus T4, a formulated PGPR product, and the arbuscular mycorrhiza fungus (AMF), Glomus intraradices. Results showed that supplementing 75% of the recommended fertilizer rate with inoculants produced plant growth, yield, and nutrient (nitrogen and phosphorus) uptake that were statistically equivalent to the full fertilizer rate without inoculants. When inoculants were used with rates of fertilizer below 75% of the recommended rate, the beneficial effects were usually not consistent; however, inoculation with the mixture of PGPR and AMF at 70% fertility consistently produced the same yield as the full fertility rate without inoculants. Without inoculants, use of fertilizer rates lower than the recommended resulted in significantly less plant growth, yield, and nutrient uptake or inconsistent impacts. The results suggest that PGPR-based inoculants can be used and should be further evaluated as components of integrated nutrient management strategies.     

Peanut (Arachis hypogaea) response to inoculation with Bradyrhizobium sp. in soils of Argentina

Soil bacteria (rhizobia) of the genus Bradyrhizobium form symbiotic relationships with peanut root cells and fix atmospheric nitrogen by converting it to nitrogenous compounds. Inoculation of peanut with rhizobia can enhance the plant’s ability to fix nitrogen from the air and thereby reduce the requirement for nitrogen fertiliser. We evaluated three Bradyrhizobium sp. strains for effect on root nodulation and on pod yield of peanut in Argentina soils, using laboratory and field experiments. Of these, strain C‐145 was the most effective in laboratory studies. In‐furrow inoculation with this strain produced increased nodule number, relative to seed inoculation. However, pod yield was not increased significantly by either type of inoculation. In view of the inconsistent response of peanut to inoculation, we examined the effect of indigenous strains of bradyrhizobia. The high degree of nodulation and nitrogen fixation produced by indigenous rhizobia were sufficient for maximal yield under the field and inoculation conditions used in this study. The data are important for future investigation of alternative inoculant strains and conditions for improving peanut production.     

Diversity of Rhizobium-Phaseolus vulgaris symbiosis: overview and perspectives

Common bean (Phaseolus vulgaris) has become a cosmopolitan crop, but was originally domesticated in the Americas and has been grown in Latin America for several thousand years. Consequently an enormous diversity of bean nodulating bacteria have developed and in the centers of origin the predominant species in bean nodules is R. etli. In some areas of Latin America, inoculation, which normally promotes nodulation and nitrogen fixation is hampered by the prevalence of native strains. Many other species in addition to R. etli have been found in bean nodules in regions where bean has been introduced. Some of these species such as R. leguminosarum bv. phaseoli, R. gallicum bv. phaseoli and R. giardinii bv. phaseoli might have arisen by acquiring the phaseoli plasmid from R. etli. Others, like R. tropici, are well adapted to acid soils and high temperatures and are good inoculants for bean under these conditions. The large number of rhizobia species capable of nodulating bean supports that bean is a promiscuous host and a diversity of bean-rhizobia interactions exists. Large ranges of dinitrogen fixing capabilities have been documented among bean cultivars and commercial beans have the lowest values among legume crops. Knowledge on bean symbiosis is still incipient but could help to improve bean biological nitrogen fixation.     

Soil nematodes mediate positive interactions between legume plants and rhizobium bacteria

Symbiosis between legume species and rhizobia results in the sequestration of atmospheric nitrogen into ammonium, and the early mechanisms involved in this symbiosis have become a model for plant-microbe interactions and thus highly amenable for agricultural applications. The working model for this interaction states that the symbiosis is the outcome of a chemical/molecular dialogue initiated by flavonoids produced by the roots of legumes and released into the soil as exudates, which specifically induce the synthesis of nodulation factors in rhizobia that initiate the nodulation process. Here, we argue that other organisms, such as the soil nematode Caenorhabditis elegans, also mediate the interaction between roots and rhizobia in a positive way, leading to nodulation. We report that C. elegans transfers the rhizobium species Sinorhizobium meliloti to the roots of the legume Medicago truncatula in response to plant-released volatiles that attract the nematode. These findings reveal a biologically-relevant and largely unknown interaction in the rhizosphere that is multitrophic and may control the initiation of the symbiosis.     

Symbiotic effectiveness of indigenous arctic rhizobia on a temperate forage legume: Sainfoin (Onobrychis viciifolia)

Sainfoin (Onobrychis viciifolia), a temperate perennial forage legume, can be nodulated by rhizobia isolated from 3 arctic legume species:Astragalus alpinus, oxytropis maydelliana andOxytropis arctobia. Arctic rhizobia, which are adapted to growth at low temperatures, may be useful in improving symbiotic nitrogen fixation during cold phases of the growing season, if they are effective on a temperate legume. In this study, we report on the symbiotic effectiveness of arctic rhizobia on sainfoin, as appraised by the total shoot dry matter yield obtained from 2 harvests. Under N-free conditions, 5 arctic strains at the first harvest and 8 at the second harvest were as effective as temperate standard strains. In the presence of 30 mgl⁻¹ NO₃-N, 7 arctic strains gave significantly higher yields than temperate strains at the second harvest. These results indicate that effective arctic rhizobia have a potential for use as inoculants on sainfoin. Contribution no 325 of Agriculture Canada Research Station a Sainte-Foy.     

Sinorhizobium fredii cultured in media supplemented with Amaranthus cruentus L. seed meal and bacterial cell survival in liquid and peat based inoculum

Nine different growth media were evaluated to determine the best growth conditions to achieve cultures of a high cell number of fast-growing rhizobia to produce inoculants. We found that Sinorhizobium fredii strains have complex nutritional requirements that were fulfilled by adding to the media 4 g Amaranthus cruentus L. seed meal/l. The survival of fast growing strains is a variable trait, but those strains that survived at high levels even after 6-month storage, hypernodulated soybeans and fixed atmospheric nitrogen at levels as high as those of Bradyrhizobium japonicum.