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.     

Polyphasic characterization of Brazilian Rhizobium tropici strains effective in fixing N2 with common bean (Phaseolus vulgaris L.)

Common bean (Phaseolus vulgaris) is native to the Americas, and Rhizobium etli is the dominant microsymbiont in both the Mesoamerican and the Andean centers of genetic diversification. Wild common beans are not found in Brazil, although the legume has been cropped in the country throughout time and all but one of the rhizobial species that nodulate it (Rhizobium gallicum) have been broadly detected in Brazilian soils. However, the majority of the effective rhizobial strains isolated so far from field-grown plants belong to R. tropici. This study describes the analysis of symbiotic and non-symbiotic genes of 15 effective R. tropici strains, isolated from four geographically distant regions in Brazil. With RFLP-PCR of the 16S and 23S rRNA genes and sequence analysis of 16S rRNA, two clusters were observed, one related to R. tropici type A and another to type B strains. Diversity in ribosomal genes was high, indicating that type A strains might represent a new species. High intraspecies diversity was also observed in the rep-PCR analysis with BOX, ERIC and REP primers. However, in the RFLP-PCR analysis of nifH and nodC genes, all R. tropici showed unique combinations of profiles, which might reflect an evolutionary strategy to maximize N₂ fixation.     

Genetic diversity of indigenous common bean (Phaseolus vulgaris) rhizobia in two Brazilian ecosystems

Although rhizobia for common bean (Phaseolus vulgaris L.) are established in most Brazilian soils, understanding of their genetic diversity is very poor. This study characterized bean strains from two contrasting ecosystems in Brazil, the Northeast Region, with a semi-arid climate and neutral soils and the South Region, with a humid subtropical climate and acid soils. Seedlings of the cultivars Negro Argel and Aporé were used to trap 243 rhizobial isolates from 12 out of 14 sites. An analysis of ERIC-PCR products revealed enormous variability, with 81% of the isolates representing unique strains considering a level of 70% of similarity. In general, there was no effect of either the bean cultivar, or the ecosystem on rhizobial diversity. One-hundred and one strains showing genetic relatedness (ERIC-PCR) less than 70% were further analyzed using restriction fragment length polymorphism (RFLP) of the 16 S rDNA cleaved with five restriction enzymes. Twenty-five different profile combinations were obtained. Rhizobium etli was the predominant species, with 73 strains showing similar RFLP profiles, while 12 other strains differed only by the profile with one restriction enzyme. Fifty strains were submitted to sequencing of a 16 S rDNA fragment, and 34 clustered with R. etli, including strains with RFLP-PCR profiles similar to those species or differing by one restriction enzyme. However, other strains differing by one or two enzymes were genetically distant from R. etli and two strains with identical profiles showed higher similarity to Sinorhizobium fredii. Other strains showed higher similarity of bases with R. tropici, R. leguminosarum and Mesorhizobium plurifarium, but some strains were quite dissimilar and may represent new species. Great variability was also verified among the sequenced strains in relation to the ability to grow in YMA at 40 °C, in LB, to synthesize melanin in vitro, as well as in symbiotic performance, including differences in relation to the described species, e.g. many R. etli strains were able to grow in LB and in YMA at 40 °C, and not all R. tropici were able to nodulate Leucaena.     

New insights into the origins and evolution of rhizobia that nodulate common bean (Phaseolus vulgaris) in Brazil

It is generally accepted that there are two major centers of genetic diversification of common beans (Phaseolus vulgaris L.): the Mesoamerican (Mexico, Colombia, Ecuador and north of Peru, probably the primary center), and the Andean (southern Peru to north of Argentina) centers. Wild common bean is not found in Brazil, but it has been grown in the country throughout recorded history. Common bean establishes symbiotic associations with a wide range of rhizobial strains and Rhizobium etli is the dominant microsymbiont at both centers of genetic diversification. In contrast, R. tropici, originally recovered from common bean in Colombia, has been found to be the dominant species nodulating field-grown common-bean plants in Brazil. However, a recent study using soil dilutions as inocula has shown surprisingly high counts of R. etli in two Brazilian ecosystems. In the present study, RFLP-PCR analyses of nodABC and nifH genes of 43 of those Brazilian R. etli strains revealed unexpected homogeneity in their banding patterns. The Brazilian R. etli strains were closely similar in 16S rRNA sequences and in nodABC and nifH RFLP-PCR profiles to the Mexican strain CFN 42^T, and were quite distinct from R. etli and R. leguminosarum strains of European origin, supporting the hypothesis that Brazilian common bean and their rhizobia are of Mesoamerican origin, and could have arrived in Brazil in pre-colonial times. R. tropici may have been introduced to Brazilian soils later, or it may be a symbiont of other indigenous legume species and, due to its tolerance to acidic soils and high temperature conditions became the predominant microsymbiont of common bean.     

Phenotypic and genotypic diversity of rhizobia in cropping areas under intensive and organic agriculture in Hungary

Nitrogen-fixing bacteria are important components of various soil–plant ecosystems and arable lands. Interrelations between the mono- or dicotyledonous crop hosts and the plant growth promoting (PGP) bacteria are highly influenced by biotic and abiotic environmental stress factors. Among the methods of community analysis available, the polymerase chain reaction (PCR)-based genetic investigations are especially important. In this study the phenotypic and genotypic diversity of rhizobia colonising white lupin (Lupinus albus L.) in cropping areas under ‘intensive’ and organic agriculture in Hungary was evaluated. Genetic diversity was assessed by DNA analyses using a BOX-PCR method. Rhizobia of the intensive agricultural practices were genetically more diverse and were also phenotypically different to the organic system. This reduced diversity can be attributed to selection pressures exerted by the re-circulation of seeds of the lupin variety harvested from the same area under organic agriculture. In addition, genetic and phenotypic differences between strains infecting the primary and secondary roots of lupin, suggest more than one initial infection event. Similar to earlier studies, our observations also highlight the importance of retaining genetic diversity in agricultural practice both of the host plants and of the rhizobial microbial populations.     

Genetic diversity and symbiotic efficiency of population of rhizobia of <Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> L. in Brazil

The genetic diversity and symbiotic efficiency among indigenous rhizobia isolates obtained from native field with or without organic fertilization and superficial mineral fertilization were investigated. Eighty-six indigenous rhizobia were isolated from these fields using four common bean varieties as trap-host. The common bean varieties Mexico 309 and Rio Tibagi selected the most efficient rhizobia strains because they showed the best yields and N contents results. The genetic characterization of 36 rhizobia isolates was evaluated by using electrophoretic profiles of amplification products using primers ERIC1-R and ERIC-2. Our results demonstrated that besides the large diversity in the indigenous rhizobial community, the genotype of the trap-host probably influences the selection of the most efficient strains.     

Effect of tillage and crop management on runoff,soil erosion and organic carbon loss

Proper management of soil organic matter is an important issue in the context of sustainable agriculture. The intensification of production and the loss of organic carbon associated with agriculture reduce the efficiency of production and the quality of the environment, especially in relation to areas exposed to erosion. The aim of this study was to determine the impact of specific tillage systems and plant cover on the organic carbon losses, as well as on runoff and soil losses, over a 6-year study period following the introduction of no-till. The first factor in the experiment was the tillage system: conventional tillage (CT) and no-till (NT). The second factor was plant cover: horse bean, spring wheat and winter oilseed rape. The results showed that runoff was 4.3 ± 0.6% higher under NT than under CT, while soil loss was 66.8 ± 2.7% lower under NT than under CT. Compared to CT, NT limited the total organic carbon losses by an average of 46.0 ± 2.9% and organic carbon bound with sediment losses by 53.2 ± 0.7%, whereas for dissolved organic carbon, there were no significant differences for the tillage systems. The anti-erosion effectiveness of NT was lower in the first year, but it increased in subsequent years after the introduction of this tillage system. Plant cover also had a significant impact on organic carbon losses and soil protection. The crops were ranked according to runoff, soil losses and organic carbon losses in the following order from lower to higher losses: winter oilseed rape > spring wheat > horse bean.     

Diversity and stress tolerance in rhizobia from Parque Chaqueño region of Argentina nodulating Prosopis alba

The aim of this work was to investigate the genetic diversity, symbiotic effectiveness, drought tolerance, and indole acetic acid production of indigenous rhizobial populations in the Parque Chaqueño of Argentina able to nodulate Prosopis alba, the dominant forest tree of this region. The populations were sampled at five locations from the Arid, Semi-arid, and Humid Chaco in the Parque Chaqueño region. A set of rhizobial strains able to nodulate P. alba was obtained and selected based on their molecular diversity. Data obtained by BOX-PCR indicated that the highest molecular variability was observed in rhizobial isolates from Semi-arid Chaco. High level of indolic compound production and tolerance to osmotic treatment were significantly (p?≤?0.05) correlated with water restrictions of the environments where the strains belonged. A small set of rhizobial strains that stimulate P. alba growth was selected from a large group of strains. The strains were identified by 16S rDNA sequencing as belonging to the genera Mesorhizobium, Bradyrhizobium, and Ensifer. To our knowledge, this is the first report of P. alba nodulation by strains other than Mesorhizobium chacoense, which was already described for the Parque Chaqueño.     

Long-term impact of no-till conservation agriculture on abundance and order diversity of soil macrofauna in continuous maize monocropping system

Unsuitable soil management in agriculture is known to results in the deterioration of soil health and the decline of biodiversity. The experiment tested whether no-tillage with mulch (NT), could boost biological activity of soil macrofauna population in continuous maize monocropping system compared with rotational tillage with mulch (RT) and conventional tillage (CT). Soil macrofauna was sampled at the end of the 2014/2015 growing season using 25?×?25?×?25?cm steel monoliths. The mean density of individual orders was higher (p?p?相似文献   

Benefits of inoculation of the common bean (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis>) crop with efficient and competitive<Emphasis Type="Italic"> Rhizobium tropici</Emphasis> strains

Cropping in low fertility soils, especially those poor in N, contributes greatly to the low common bean (Phaseolus vulgaris L.) yield, and therefore the benefits of biological nitrogen fixation must be intensively explored to increase yields at a low cost. Six field experiments were performed in oxisols of Paraná State, southern Brazil, with a high population of indigenous common bean rhizobia, estimated at a minimum of 10³ cells g^–1 soil. Despite the high population, inoculation allowed an increase in rhizobial population and in nodule occupancy, and further increases were obtained with reinoculation in the following seasons. Thus, considering the treatments inoculated with the most effective strains (H 12, H 20, PRF 81 and CIAT 899), nodule occupancy increased from an average of 28% in the first experiment to 56% after four inoculation procedures. The establishment of the selected strains increased nodulation, N₂ fixation rates (evaluated by total N and N-ureide) and on average for the six experiments the strains H 12 and H 20 showed increases of 437 and 465 kg ha^–1, respectively,in relation to the indigenous rhizobial population. A synergistic effect between low levels of N fertilizer and inoculation with superior strains was also observed, resulting in yield increases in two other experiments. The soil rhizobial population decreased 1 year after the last cropping, but remained high in the plots that had been inoculated. DGGE analysis of soil extracts showed that the massive inoculation apparently did not affect the composition of the bacterial community.     

INTERACTIONS BETWEEN COMMON BEAN GENOTYPES AND RHIZOBIA STRAINS ISOLATED FROM MOROCCAN SOILS FOR GROWTH,PHOSPHATASE AND PHYTASE ACTIVITIES UNDER PHOSPHORUS DEFICIENCY CONDITIONS

The improvement of common bean production requires the selection of effective rhizobia strains and Phaseolus vulgaris genotypes adapted to available soil phosphorus limitations. The interactions between bean genotypes and rhizobia were studied in hydroponic culture using six genotypes and four strains, CIAT899 as reference and three strains isolated from nodule of farmer's fields in the Marrakech region. The phosphorus (P) sub-deficiency caused a significant reduction on shoot biomass in some bean genotype-rhizobia combinations. Nodule biomass is significantly more reduced under P limitation for several combinations tested. Bean plants inoculated with these local rhizobial strains showed higher nodulation and an increase of nodules phytase and phosphatase activities under phosphorus sub-deficiency especially for RhM11 strain. It was concluded that the studied bean-rhizobia symbiosis differ in their adaptation to phosphorus sub-deficiency and the nodule phosphatases and phytases activities may constitute a strategy of nodulated bean plants to adapt their nitrogen fixation to P deficiency.     

Organic farming fosters agroecosystem functioning in Argentinian temperate soils: Evidence from litter decomposition and soil fauna

Benefits of organic farming on soil fauna have been widely observed and this has led to consider organic farming as a potential approach to reduce the environmental impact of conventional agriculture. However, there is still little evidence from field conditions about direct benefits of organic agriculture on soil ecosystem functioning. Hence, the aims of this study were to compare the effect of organic farming versus conventional farming on litter decomposition and to study how this process is affected by soil meso- and macrofauna abundances. Systems studied were: (1) organic farming with conventional tillage (ORG), (2) conventional farming with conventional tillage (CT), (3) conventional farming under no-tillage (NT), and (4) natural grassland as control system (GR). Decomposition was determined under field conditions by measuring weight loss in litterbags. Soil meso- and macrofauna contribution on decomposition was evaluated both by different mesh sizes and by assessing their abundances in the soil. Litter decomposition was always significantly higher after 9 and 12 months in ORG than in CT and NT (from 2 to 5 times in average), regardless decomposer community composition and litter type. Besides, mesofauna, macrofauna and earthworm abundances were significantly higher in ORG than in NT and CT (from 1.6 to 3.8, 1.7 to 2.3 and 16 to 25 times in average, respectively for each group). These results are especially relevant firstly because the positive effect of ORG in a key soil process has been proved under field conditions, being the first direct evidence that organic farming enhances the decomposition process. And secondly because the extensive organic system analyzed here did not include several practices which have been recognized as particularly positive for soil biota (e.g. manure use, low tillage intensity and high crop diversity). So, this research suggests that even when those practices are not applied, the non-use of agrochemicals is enough to produce positive changes in soil fauna and so in decomposition dynamics. Therefore, the adoption of organic system in an extensive way can also be suggested to farmers in order to improve ecosystem functioning and consequently to achieve better soil conditions for crop production.     

Diversity of <Emphasis Type="Italic">Trifolium ambiguum</Emphasis>—nodulating rhizobia from the lower Caucasus

Kura clover (Trifolium ambiguum M.B.) is a perennial rhizomatous forage legume whose use is currently limited by difficulties in its establishment in part attributable to nodulation problems and very specific rhizobial requirements. A limited number of Kura clover-nodulating rhizobial strains are currently available and many have a limited effectiveness. In this study, 128 rhizobia were isolated from four sites in the center of origin of Kura clover (i.e., two in Azerbaijan, one in Armenia, and one in Northwest Iran) using the three ploidy levels of Kura clover (diploid, tetraploid, and hexaploid), red clover (Trifolium pratense L.), and white clover (Trifolium repens L.) plants as trap hosts. Rhizobia were fingerprinted using repetitive extragenic palindromic polymerase chain reaction (BOXA1R primer) and their genetic diversity was measured using the Shannon-Weaver diversity index. The nodulation specificity and phenotypic diversity of a subset of 13 isolates was determined. Genetic diversity among the 128 isolates was large and similar for rhizobia grouped according to their geographic origin or original host plant. Phenotypic diversity was significant; percentage of similarity among 13 isolates ranging between 38 and 92%. Nodulation specificity of the Kura clover-nodulating rhizobial isolates studied was less complex and not as clearly delineated as previously reported. Some strains originally isolated from Kura clover could effectively nodulate more than one ploidy level of Kura clover and even one or both of two other Trifolium species (i.e., red clover and white clover). Three strains formed effective nodules on both Kura clover and white clover; however, none promoted plant growth of both species to levels currently obtained with commercial inoculants when evaluated in a growth chamber. Rhizobial isolates that are highly effective with both species have yet to be identified.     

Genetic diversity analysis in Phaseolus vulgaris L. using morphological traits

Common bean (Phaseolus vulgaris) is an important export crop in Kyrgyzstan since the end of the twentieth century. Genetic diversity analysis of common bean populations is useful for breeding programs, as it helps to select genetic material to be used for further crossing. Twenty-seven common bean accessions were analyzed using 13 qualitative morphological traits. In some cases, obtained morphological data were combined with previously published results based on microsatellite markers. The similarity matrices generated from the molecular and morphological data were significantly correlated (r = 0.49, P < 0.01). Cluster analyses based on Dice’s similarity coefficient were constructed based on morphological data and the combined data set of morphology and microsatellite, and both grouped the 27 accessions according to their origin: 15 belonged to the Andean and 12 to the Mesoamerican gene pool. On average, the Andean accessions were less diverse than the Mesoamerican accessions. The average diversity based on the Shannon diversity index for the 13 qualitative morphological traits was 0.05. Overall, this study revealed that qualitative morphological markers are efficient in assigning modern cultivars to their gene pools of origin.     

Soil sustainability indicators following conservation tillage practices under subtropical maize and bean crops

Conservation management systems such as no tillage may enhance sequestration of soil C. The soil properties that contribute to soil C storage under such systems are still largely unknown, especially in subtropical agroecosystems. We investigated the influence of tillage [mouldboard plough (MP) and no tillage (NT)] on soil organic C, microbial biomass and activity, structural stability and mycorrhizal status of a field cultivated with maize (Zea mays L.) or bean (Phaseolus vulgaris L.) on a Vertisol in Northern Tamaulipas, Mexico. Crop type, tillage system and soil depth had a significant effect on soil organic C, aggregate stability and bulk density. Soil organic C, microbial biomass C and N and dehydrogenase and phosphatase activities were greater with NT than with MP, particularly under bean cultivation. In the 0–5 cm layer, microbial biomass C and N were, on average, about 87 and 51% greater in the soils cultivated with bean and maize, respectively, under NT than under MP. Higher levels of mycorrhizal propagules, glomalin related soil protein (GRSP) and stable aggregates were produced under NT than under MP in both crops. The no-tillage system can be considered an effective management practice for carrying out sustainable agriculture under subtropical conditions, due to its improvement of soil physical and biochemical quality and soil C sequestration.     

农业系统中生物多样性利用的研究现状与未来思考

"现代农业"生产力高,但生物多样性简单化、生物之间的相互作用及其生态学效应常常被忽略,其生产力的稳定性主要依赖于化学肥料、农药、灌溉和高产品种等的投入。传统农业则是利用当地生物多样性(物种多样性和遗传多样性)和生物之间的相互作用来产出食物和维持系统的稳定。因而现代农业能否借鉴传统农业对生物多样性利用的经验,将工业化模式的现代农业转换为生物多样性利用与现代技术相结合的农业受到关注。本文分析了农业系统中生物多样性的特点及农业方式对农业生物多样性的影响;综述了农业系统中生物多样性利用模式与效应方面的研究进展;讨论了在现代农业系统中,利用生物多样性需要开展的研究,即区域上如何布局农业景观多样性,农田内如何根据生物之间的互惠关系配置物种多样性的种养体系,如何建设与生物多样性利用相应的田间设施和发展新型的农业机械、并建立以信息化为基础的管理体系。     

科尔沁沙地土地利用变化对大型土壤节肢动物群落影响

土地利用类型是地球表层系统最突出的景观标志,不仅可以客观地展示地球表面特征空间格局的活动,还反映着地球表面景观的时空动态过程[1-2].由于土地利用变化与人类活动和全球气候变化密切相关,对生物多样性消长、生态环境演变、生态安全水平以及人类可持续性发展有着重要影响,因而其研究已成为当今全球变化研究的前沿和热点课题[1,3].     

Long-term tillage and crop rotation effects on microbial biomass and C and N mineralization in a Brazilian Oxisol

Crop rotation and tillage impact microbial C dynamics, which are important for sequestering C to offset global climate change and to promote sustainable crop production. Little information is available for these processes in tropical/subtropical agroecosystems, which cover vast areas of terrestrial ecosystems. Consequently, a study of crop rotation in combination with no tillage (NT) and conventional tillage (CT) systems was conducted on an Oxisol (Typic Haplorthox) in an experiment established in 1976 at Londrina, Brazil. Soil samples were taken at 0–50, 50–100 and 100–200 mm depths in August 1997 and 1998 and evaluated for microbial biomass carbon (MBC) and mineralizable C and N. There were few differences due to crop rotation, however there were significant differences due to tillage. No tillage systems increased total C by 45%, microbial biomass by 83% and MBC:total C ratio by 23% at 0–50 mm depth over CT. C and N mineralization increased 74% with NT compared to CT systems for the 0–200 mm depth. Under NT, the metabolic quotient (CO₂ evolved per unit of MBC) decreased by 32% averaged across soil depths, which suggests CT produced a microbial pool that was more metabolically active than under NT systems. These soil microbial properties were shown to be sensitive indicators of long-term tillage management under tropical conditions.     

Genetic diversity in accessions of lima bean (Phaseolus lunatus L.) determined from agro-morphological descriptors and SSR markers for use in breeding programs in Brazil

Genetic Resources and Crop Evolution - Lima bean (Phaseolus lunatus L.) have high genetic diversity, especially in Brazil, which has been maintained both on farms and in germplasm banks. Knowledge...     

Impact of tillage and crop rotation on light fraction and intra-aggregate soil organic matter in two Oxisols

It is well known that no-tillage (NT) practices can promote greater stocks of soil organic matter (SOM) in the soil surface layer compared to conventional tillage (CT) by enhancing the physical protection of aggregate-associated C in temperate soils. However, this link between tillage, aggregation and SOM is less well established for tropical soils, such as Oxisols. The objective of this study was to investigate the underlying mechanisms of SOM stabilization in Oxisols as affected by different crop rotations and tillage regimes at two sites in southern Brazil. Soils were sampled from two agricultural experiment sites (Passo Fundo and Londrina) in southern Brazil, with treatments comparing different crop rotations under NT and CT management, and a reference soil under native vegetation (NV). Free light fraction (LF) and intra-aggregate particulate organic matter (iPOM) were isolated from slaking-resistant aggregates. Of the total C associated with aggregates, 79–90% was found in the mineral fraction, but there were no differences between NT and CT. In contrast, tillage drastically decreased LF-C concentrations in the 0–5 cm depth layer at both sites. In the same depth layer of NT systems at Londrina, the concentrations of iPOM-C were greater when a legume cover crop was included in the rotation. At Londrina, the order of total iPOM-C levels was generally NV > NT > CT in the 0–5 cm depth interval, but the difference between NT and CT was much less than in Passo Fundo. At Passo Fundo, the greatest concentrations and differences in concentrations across tillage treatments were found in the fine (53–250 μm) iPOM fractions occluded within microaggregates. In conclusion, even though no aggregate hierarchy exists in these Oxisols, our results corroborate the concept of a stabilization of POM-C within microaggregates in no-tillage systems, especially when green manures are included in the rotation.