Differences in common bean rhizobial populations associated with soil tillage management in southern Brazil

Progressive adoption of no-tillage (NT) agriculture in the tropics is finally reversing physical, chemical, and biological erosion of soil and in Brazil, an estimated 19 Mha are now devoted to NT. Common bean (Phaseolus vulgaris L.) is a main component of Brazilian agriculture, and enhancement of yields has been achieved under NT as a result of mitigation of environmental stresses, resulting in higher N₂ fixation. However, the effects of NT on rhizobial diversity are poorly understood. This study evaluated rhizobial diversity in soils planted to common bean under NT or conventional tillage (CT) systems that were compared with natural grassland used for grazing, in the State of Santa Catarina, southern Brazil. Genetic diversity was assessed by the amplification of the DNA by PCR with specific primers (BOX-PCR) and by RFLP-PCR analyses of the 16S rDNA region. A high level of diversity was observed among strains from all three systems, such that the similarity in the clustering analysis of BOX-PCR products ranged from 36% under natural grassland to only 23% for CT strains. High polymorphism was confirmed in the RFLP-PCR analysis; forty-seven different profiles were obtained, none sharing high similarity with the profiles of reference species of common bean rhizobia. These results indicate that other tropical rhizobial species remain to be described. Genetic diversity was higher among the NT than the CT rhizobial strains, especially when the RFLP-PCR profiles were considered. Genetic diversity in the natural grassland was lower than in the cropped systems, possibly due to absence of the host plant and stubble burning in winter. Average yields in the area under NT (e.g. common bean, approximately 1500 kg ha⁻¹) have been about 30% higher than under CT, therefore high rhizobial diversity may be a parameter indicative of superior soil quality.     

Soil microbial activity and crop sustainability in a long-term experiment with three soil-tillage and two crop-rotation systems

Reduction in soil disturbance can stimulate soil microbial biomass and improve its metabolic efficiency, resulting in better soil quality, which in turn, can increase crop productivity. In this study we evaluated microbial biomass of C (MB-C) by the fumigation-extraction (FE) or fumigation-incubation (FI) method; microbial biomass of N (MB-N); basal respiration (BR) induced or not with sucrose; metabolic quotient (obtained by the ratio BR/MB-C) induced (qCO₂(S)), or not with sucrose (qCO₂); and crop productivity in a 14-year experiment in the state of Paraná, southern Brazil. The experiment consisted of three soil-tillage systems [no-tillage (NT), conventional tillage (CT) and no-tillage using a field cultivator every 3 years (FC)] and two cropping systems [a soybean–wheat-crop sequence (CS), and a soybean–wheat–white lupin–maize–black oat–radish crop rotation (CR)]. There were six samplings in the 14th year, starting at the end of the winter crop (wheat in the CS and lupin in the CR plots) and finishing at full flowering of the summer crop (soybean in the CS and maize in the CR). Differences in microbiological parameters were greater than those detected in the total C (TCS) and total N (TNS) contents of the soil organic matter (SOM). Major differences were attributed to tillage, and on average NT was higher than the CT in the following parameters: TCS (19%), TNS (21%), MB-C evaluated by FE (74%) and FI (107%), and MB-N (142%). The sensibility of the microbial community and processes to soil disturbance in the tropics was highlighted, as even a moderate soil disturbance every 3 years (FC) affected microbial parameters but not SOM. The BR was the parameter that most promptly responded to soil disturbance, and strong differences were perceived by the ratio of qCO₂ evaluated with samples induced and non-induced with sucrose. At plowing, the qCO₂(S):qCO₂ was five times higher under CT, indicating a C-starving low-effective microbial population in the C-usage. In general, crop rotation had no effect on microbial parameters or SOM. Grain yield was affected by tillage and N was identified as a limiting nutrient. Linear regressions between grain yields and microbial parameters showed that soybean was benefited from improvements in the microbial biomass and metabolic efficiency, but with no significant effects observed for the maize crop. The results also indicate that the turnover of C and N in microbial communities in tropical soils is rapid, reinforcing the need to minimize soil disturbance and to balance inputs of N and C.     

Sampling effects on the assessment of genetic diversity of rhizobia associated with soybean and common bean

Biological nitrogen fixation plays a key role in agriculture sustainability, and assessment of rhizobial diversity contributes to worldwide knowledge of biodiversity of soil microorganisms, to the usefulness of rhizobial collections and to the establishment of long-term strategies aimed at increasing contributions of legume-fixed N to agriculture. Although in recent decades the use of molecular techniques has contributed greatly to enhancing knowledge of rhizobial diversity, concerns remain over simple issues such as the effects of sampling on estimates of diversity. In this study, rhizobia were isolated from nodules of plants grown under field conditions, in pots containing soil, or in Leonard jars receiving a 10⁻² or a 10⁻⁴ serially-diluted soil inoculum, using one exotic (soybean, Glycine max) and one indigenous (common bean, Phaseolus vulgaris) legume species. The experiments were performed using an oxisol with a high population (10⁵ cells g⁻¹ soil) of both soybean rhizobia, composed of naturalized strains introduced in inoculants and of indigenous common-bean rhizobia. BOX-PCR was used to evaluate strain diversity, while RFLP-PCR of the ITS (internally transcribed spacer) region with five restriction enzymes aimed at discriminating rhizobial species. In both analyses the genetic diversity of common-bean rhizobia was greater than that of soybean. For the common bean, diversity was greatly enhanced at the 10⁻⁴ dilution, while for the soybean dilution decreased diversity. Qualitative differences were also observed, as the DNA profiles differed for each treatment in both host plants. Differences obtained can be attributed to dissimilarity in the history of the introduction of both the host plant and the rhizobia (exotic vs. indigenous), to host-plant specificity, rhizobial competitiveness, and population structure, including ease with which some types are released from microcolonies in soil. Therefore, sampling method should be considered both in the interpretation and comparison of the results obtained in different studies, and in the setting of the goals of any study, e.g. selection of competitive strains, or collection of a larger spectrum of rhizobia. Furthermore, effects of sampling should be investigated for each symbiosis.     

Three decades of soil microbial biomass studies in Brazilian ecosystems: Lessons learned about soil quality and indications for improving sustainability

Soil microbial biomass plays important roles in nutrient cycling, plant-pathogen suppression, decomposition of residues and degradation of pollutants; therefore, it is often regarded as a good indicator of soil quality. We reviewed more than a hundred studies in which microbial biomass-C (MB-C), microbial quotient (MB-C/TSOC, total soil organic carbon) and metabolic quotient (qCO₂) were evaluated with the objective of understanding MB-C responses to various soil-management practices in Brazilian ecosystems. These practices included tillage systems, crop rotations, pastures, organic farming, inputs of industrial residues and urban sewage sludge, applications of agrochemicals and burning. With a meta-analysis of 233 data points, we confirmed the benefits of no-tillage in preserving MB-C and reducing qCO₂ in comparison to conventional tillage. A large number of studies described increases in MB-C and MB-C/TSOC due to permanent organic farming, also benefits from crop rotations particularly with several species involved, whereas application of agrochemicals and burning severely disturbed soil microbial communities. The MB-C decreased in overgrazed pastures, but increased in pastures rotated with well-managed crops. Responses of MB-C, MB-C/TSOC and qCO₂ to amendment with organic industrial residues varied with residue type, dose applied and soil texture. In conclusion, MB-C and related parameters were, indeed, useful indicators of soil quality in various Brazilian ecosystems. However, direct relationships between MB-C and nutrient-cycling dynamics, microbial diversity and functionality are still unclear. Further studies are needed to develop strategies to maximize beneficial effects of microbial communities on soil fertility and crop productivity.     

Physical,chemical and microbiological soil attributes influence soil greenhouse gases fluxes in Atlantic Forest and pine (Pinus taeda) plantations in Brazil

Forest soils can be sources or sinks of greenhouse gases (GHGs) depending on soil attributes that affect biomass and activity of soil micro-organisms involved in GHGs fluxes. In this work, we tested the hypothesis that soil physical, chemical and microbiological attributes, under different forests ecosystems, affect the soil GHGs [nitrous oxide (N₂O), carbon dioxide (CO₂) and methane (CH₄)] fluxes. The study was carried out in two locations in southern Brazil in 2019, with three experimental plots of 900 m² in native forests of the Atlantic Forest biome and in loblolly pine (Pinus taeda) plantations. Air samples released from the soil surface were analysed for concentration and flux of CO₂, N₂O and CH₄. Soil samples were analysed for chemical attributes, density (Ds), soil microporosity (MiPs), soil macroporosity (MaPs), total porosity (TP), water-filled pore space (WFPS), microbial biomass carbon (MB-C), basal respiration (BR), microbial (qMic) and metabolic (qCO₂) quotient and activities of soil urease and β-glucosidase enzymes. The seasons influenced the CO₂ and N₂O emissions, probably because of the changes in seasonal conditions. However, native forests consumed more CH₄ than pine plantations. Meanwhile, the native forests presented soils with lower Ds (average 21.5% lower), more TP (average 12.5% higher) and more moisture (average 33% higher), which improved the microbiological attributes of the soil (20% to 60% more MB-C, 67% higher urease activity and 30% higher β-glucosidase activity) compared with pine plantations. Native forests contributed more intensely to CH₄ consumption than pine plantations because they present better physical, chemical and microbiological soil conditions. Therefore, it is possible that forestry practices that improve soil physical attributes are likely to contribute to increase CH₄ consumption, and to reduce GHGs emissions in forest ecosystems.     

Are the rates of photosynthesis stimulated by the carbon sink strength of rhizobial and arbuscular mycorrhizal symbioses?

Rhizobial and arbuscular mycorrhizal (AM) symbioses each may consume 4-16% of recently photosynthetically-fixed carbon to maintain their growth, activity and reserves. Rhizobia and AM fungi improve plant photosynthesis through N and P acquisition, but increased nutrient uptake by these symbionts does not fully explain observed increases in the rate of photosynthesis of symbiotic plants. In this paper, we test the hypothesis that carbon sink strength of rhizobial and AM symbioses stimulates the rates of photosynthesis. Nutrient-independent effects of rhizobial and AM symbioses result in direct compensation of C costs at the source. We calculated the response ratios of photosynthesis and nutrient mass fraction in the leaves of legumes inoculated with rhizobial and/or AM fungi relative to non-inoculated plants in a number of published studies. On average, photosynthetic rates were significantly increased by 28 and 14% due to rhizobial and AM symbioses, respectively, and 51% due to dual symbiosis. The leaf P mass fraction was increased significantly by 13% due to rhizobial symbioses. Although the increases were not significant, AM symbioses increased leaf P mass fraction by 6% and dual symbioses by 41%. The leaf N mass fraction was not significantly affected by any of the rhizobial, AM and dual symbioses. The rate of photosynthesis increased substantially more than the C costs of the rhizobial and AM symbioses. The inoculation of legumes with rhizobia and/or AM fungi, which resulted in sink stimulation of photosynthesis, improved the photosynthetic nutrient use efficiency and the proportion of seed yield in relation to the total plant biomass (harvest index). Sink stimulation represent an adaptation mechanism that allows legumes to take advantage of nutrient supply from their microsymbionts without compromising the total amount of photosynthates available for plant growth.     

Responses of legumes to rhizobia and arbuscular mycorrhizal fungi: A meta-analysis of potential photosynthate limitation of symbioses

Legumes are prized for their seed protein and lipid mass fractions. Since legumes spend up to 4-16% of photosynthesis on each of the rhizobial and arbuscular mycorrhizal (AM) fungal symbioses, it might be expected that positive responses in yield due to rhizobial and AM symbioses are accompanied by decreases in seed protein and lipid mass fractions due to a photosynthate (C) limitation. We performed a meta-analysis of 348 data points from published studies with 12 legume species to test whether yield, harvest index, and seed protein and lipid mass fractions are affected by symbioses. There was a significant increase in yield due to rhizobial inoculation (16% in the field; 59% in pot experiments). There were no responses of yield to AM fungi and rhizobial + AM fungi inoculations in the field (presumably because an AM fungi-free control cannot be ensured), but significant responses in pots (45% with AM fungi; 44% with rhizobial + AM fungi). Rhizobial inoculation improved seed protein mass fraction by 7% in the field; AM fungi increased this parameter by 14% in pots. There were no discernable effects of symbioses on seed lipid mass fraction. Rhizobial symbioses in the field increased harvest index (+5%), but AM fungi did not affect harvest index. In conclusion, increases in yield due to symbioses also resulted in increases in seed protein and constant lipid mass fractions, indicating that legumes are not C-limited under symbiotic conditions.     

Soybean [<Emphasis Type="Italic">Glycine max</Emphasis> (L.) Merrill] rhizobial diversity in Brazilian oxisols under various soil,cropping, and inoculation managements

In this study, soybean nodules were collected from 12 sites in the State of Mato Grosso, in the Brazilian Cerrados, where both exotic soybean [Glycine max (L.) Merrill] and bradyrhizobial strains have been introduced from 1 to 18 years before. All soils were originally devoid of rhizobia capable of effectively nodulating soybean and varied in terms of chemical and physical properties, inoculation procedures, and cropping systems. Rhizobial genetic diversity was assessed on 240 isolates by rep-PCR fingerprinting with BOX primer, and indices of diversity (abundance-based coverage estimator and traditional and modified Shannon indices) were applied to the profiles obtained. The genetic diversity was much greater than expected, as after the introduction of a maximum of four strains, up to 13 profiles were identified, some sharing many similar bands with the inoculant strains, but others quite distinct from the putative parental genotypes. The increase in the number of rep-PCR profiles could be attributed to genetic variability due to the stressful tropical environmental conditions, but also might indicate that indigenous rhizobia become capable of nodulating the host legume. After the third year of cropping with the host legume, inoculation did not affect rhizobial diversity. A high content of clay decreased diversity in comparison with that seen in a sandy soil, probably due to reduced aeration. Diversity was higher under the no-tillage system when compared to the conventional tillage management, highlighting the importance of maintaining crop residues in tropical soils. Understanding the ecology of exotic rhizobia after being introduced into new cropping areas represents a first step towards the establishment of better strategies of inoculation, which in turn may result in sustainability and higher plant yields.