Cécile Revellin

Trends in rhizobial inoculant production and use

Rhizobia inoculants have contributed to increase N2 fixation and yield in legumes crops. However, most of the inoculants produced world-wide are of poor or suboptimal quality. We discuss here why some of them are poor products and how to improve their quality and efficacy. Reported data on the inoculation rate effect can be used to design good inoculants. Technologies are now available to produce inoculants with a shelf-life of more than 1 year. Available quality control methods can help to improve the quality of inoculants although they do not take into account the physiological satus of the rhizobia. Unfortunately quality control is not commonly used except in major inoculant companies and the quality of inoculants sold on the market is low. The need for an increase in quality standards is discussed especially for the number of rhizobia delivered per seed and for the presence of contaminants. Some new technologies which able to increase efficacy and reliability of inoculation are discussed.

Effect of some fungicide seed treatments on the survival of Bradyrhizobium japonicum and on the nodulation and yield of soybean [Glycine max. (L) Merr.]

Several commercial fungicide seed treatments were evaluated for their possible effect on the survival of Bradyrhizobium japonicum on seeds and on the nodulation and yield of soybeans in a greenhouse and a field experiment. quinolate Pro (carbendazim and oxine copper), Vitavax 200FF (carboxin and thiram), and Monceren (pencycuron) had a small effect or no effect on the survival of B. japonicum and on the nodulation and yield of soybeans. They can thus be considered compatible with soybean seed inoculation. Germipro UFB (carbendazim and iprodione), Apron 35J (metalaxyl), and Tachigaren (hymexazol) decreased B. japonicum survival and the nodulation and yield of soybeans and thus cannot be considered compatible with soybean seed inoculation.

Evidence for homoploid speciation in Phytophthora alni supports taxonomic reclassification in this species complex.

Alder decline has been a problem along European watercourses since the early 1990s. Hybridization was identified as the main cause of this emerging disease. Indeed, the causal agent, a soil-borne pathogen named Phytophthora alni subsp. alni (Paa) is the result of interspecific hybridization between two taxa, Phytophthora alni subsp. multiformis (Pam) and Phytophthora alni subsp. uniformis (Pau), initially identified as subspecies of Paa. The aim of this work was to characterize the ploidy level within the P. alni complex that is presently poorly understood. For that, we used two complementary approaches for a set of 31 isolates of Paa, Pam and Pau: (i) quantification of allele copy number of three single-copy nuclear genes using allele-specific real-time PCR and (ii) comparison of the genome size estimated by flow cytometry. Relative quantification of alleles of the three single-copy genes showed that the copy number of a given allele in Paa was systematically half that of its parents Pau or Pam. Moreover, DNA content estimated by flow cytometry in Paa was equal to half the sum of those in Pam and Pau. Our results therefore suggest that the hybrid Paa is an allotriploid species, containing half of the genome of each of its parents Pam and Pau, which in turn are considered to be allotetraploid and diploid, respectively. Paa thus results from a homoploid speciation process. Based on published data and on results from this study, a new formal taxonomic name is proposed for the three taxa Paa, Pam and Pau which are raised to species status and renamed P. ×alni, P. ×multiformis and P. uniformis, respectively.

Changes in the physiological and agricultural characteristics of peat-based Bradyrhizobium japonicum inoculants after long-term storage.

Abstract Commercial soybean inoculants processed with sterilised peat and stored at 20 °C for 1–8 years were used as experimental materials to assess the changes in the physiological activity of Bradyrhizobium japonicum after storage. Viable counts decreased and physiological characteristics of the bacterium changed during storage, with an increase in the time taken for colony appearance on a medium without yeast extract, an increase in the lag time for nodule appearance on soybean grown in glass tubes and a decrease in survival on seeds. All the inoculants produced a significant increase in grain yield in a field experiment. The percentage of efficient cells in the field (relative to the plate counts) decreased as the length of storage increased. These results suggest that the physiological activity of B. japonicum cells changes after storage. Practical implications for inoculant quality control are discussed.

Understanding and managing soil biodiversity: a major challenge in agroecology

Soils are living environments in which particularly abundant and diverse microbiome and fauna are evolving. The resulting biological functioning has a direct impact not only on soil fertility but also on a series of ecosystems services. Thus, microbial communities are involved in geochemical cycles in which microbial enzymes catalyse the different steps. Modulation of the corresponding activities is essential as these affect plant growth and environmental quality. In general, biodiversity affects both the productivity and stability of agroecosystems. It is therefore of paramount importance to take soil biodiversity and biological functioning into account when designing cropping systems and evaluating their impacts. The progress achieved in soil microbiology in recent years now makes it possible to propose analyses of soil biology, as has been feasible for many years for soil physicochemistry. These analyses obviously require the use of standardized procedures for soil sampling, measuring the abundance and diversity of the microbial communities, as well as the identification of bioindicators. Similarly, referential systems need to be established to interpret these analyses and diagnose the biological status of soils, and, more especially, to determine whether the obtained values are within the range of variations normal for a given soil type and land use. Great progress to standardize such procedures and establish referential systems has been achieved during large-scale research programmes carried out to characterize biodiversity on national and European scales. These diagnostic elements need to be accompanied by recommendations. The aim of ongoing research is thus to propose aids for decision-making, based on the results of biological analyses, so attempts can be made to monitor and manage biodiversity to satisfy soil fertility requirements and ensure the ecosystem services expected of soils.

Inoculants of leguminous crops for mitigating soil emissions of the greenhouse gas nitrous oxide

Nitrous oxide (N2O) is a greenhouse gas which is also responsible for ozone depletion, that mainly originates from soils and agricultural activities. We investigated the ability of inoculants of Bradyrhizobium japonicum carrying the nosZ gene to mitigate soil N2O emissions. The consumption of N2O by strains of Bradyrhizobium japonicum (USDA110 and MSDJ G49) was investigated both on inoculated soybean plants cultivated in soil pots during a greenhouse experiment and on detached nodules submitted to gradients of oxygen and N2O concentrations in laboratory conditions. During the greenhouse experiment, we switched from a system acting as an N2O source (soil + soybean inoculated with a nosZ gene depleted strain) to a system acting as an N2O sink (soil + soybean inoculated with strains carrying the nosZ gene). Nodules of Bradyrhizobium japonicum USDA110 and MSDJ G49 were both able to reduce N2O under aerobic conditions at rates increasing with N2O concentrations. Calculations using the obtained quantitative results clearly suggest an environmental benefit of this process on the field scale. This study demonstrates that the inoculation of rhizobia strains on leguminous crops is a promising area for mitigating N2O emission by cultivated soils and that further researches are required to best evaluate quantitative benefits.

Improved enumeration of Bradyrhizobium japonicum in commercial soybean inoculants using selective media

Two selective media proposed for the enumeration of Bradyrhizobium japonicum were tested using six strains of different origin and eight different commercial soybean inoculants. These media contained tetracycline, rifampicin and chloramphenicol to control bacterial contaminants, and cycloheximide and pimafucin to control fungal contaminants. They were compared with previously described selective media and plant infection technique counts. The proposed media provided better control of contaminants than previously described media, gave counts of B. japonicum similar to those obtained by the plant infection technique, and so may be used for quality control of commercial inoculants.

Repeated sequence RSα is diagnostic for Bradyhizobium japonicum and Bradyrhizobium elkanii

The genome of Brahyrhizobium japonicum and B. elkanii contains multiple copies of the repeated DNA sequence RSα. A collection of 18 B. japonicum, 4 B. elkanii and 72 other bacterial strains was screened by polymerase chain reaction (PCR) using a pair of primers specific for RSα. Only strains of B. japonicum and B. elkanii gave the predicted amplification product. Restriction analysis of PCR products obtained from different strains of B. japonicum showed that the RSα sequence was generally conserved. The usefulness of RSα as a specific probe for Brahyrhizobium strains capable of nodulating soybean was also demonstrated.

Rapeseed-legume intercrops: plant growth and nitrogen balance in early stages of growth and development

In this study we tested whether legumes can improve the growth and N and S nutrition of rapeseed in an intercropping system and compared the effect of mixtures on legume N-fixation and soil N-resources. Rapeseed was cultivated in low N conditions in monocrops using one (R) or two plants (RR) per pot and in mixtures with lupine, clover or vetch. The R monocrop was the most relevant control, intraspecific competition inducing a significant growth delay resulting in a significantly lower leaf number, in RR monocrop compared to R and the three mixtures considered. Plant biomass, and the N and S contents of rapeseed grown in mixtures were the same than those measured in R monocrop. Compared to the monocrop, the proportion of N derived from the atmosphere was increased by 34, 140 and 290% in lupine, clover and vetch, respectively when intercropped with rapeseed. In mixture with clover and lupine, the soil N pool at harvest was higher than in other treatments, while N export by crop was constant. Legumes suffered from competition for soil S resulting in a decrease of 40% in their S content compared to the monocrop. Compared to rapeseeds grown in R monocrop and in mixture with lupine and vetch, rapeseed mixed with clover showed significantly higher SPAD values in old leaves. In our conditions, mixing legumes with rapeseed is relevant to reduce N fertilization and improve nutrition and growth of rapeseed.

Long-Term Exposure of Agricultural Soil to Veterinary Antibiotics Changes the Population Structure of Symbiotic Nitrogen-Fixing Rhizobacteria Occupying Nodules of Soybeans (Glycine max)

ABSTRACT Antibiotics are entrained in agricultural soil through the application of manures from medicated animals. In the present study, a series of small field plots was established in 1999 that receive annual spring applications of a mixture of tylosin, sulfamethazine, and chlortetracycline at concentrations ranging from 0.1 to 10 mg · kg−1 soil. These antibiotics are commonly used in commercial swine production. The field plots were cropped continuously for soybeans, and in 2012, after 14 annual antibiotic applications, the nodules from soybean roots were sampled and the occupying bradyrhizobia were characterized. Nodules and isolates were serotyped, and isolates were distinguished using 16S rRNA gene and 16S to 23S rRNA gene intergenic spacer region sequencing, multilocus sequence typing, and RSα fingerprinting. Treatment with the antibiotic mixture skewed the population of bradyrhizobia dominating the nodule occupancy, with a significantly larger proportion of Bradyrhizobium liaoningense organisms even at the lowest dose of 0.1 mg · kg−1 soil. Likewise, all doses of antibiotics altered the distribution of RSα fingerprint types. Bradyrhizobia were phenotypically evaluated for their sensitivity to the antibiotics, and there was no association between in situ treatment and a decreased sensitivity to the drugs. Overall, long-term exposure to the antibiotic mixture altered the composition of bradyrhizobial populations occupying nitrogen-fixing nodules, apparently through an indirect effect not associated with the sensitivity to the drugs. Further work evaluating agronomic impacts is warranted. IMPORTANCE Antibiotics are entrained in agricultural soil through the application of animal or human waste or by irrigation with reused wastewater. Soybeans obtain nitrogen through symbiotic nitrogen fixation. Here, we evaluated the impact of 14 annual exposures to antibiotics commonly used in swine production on the distribution of bradyrhizobia occupying nitrogen-fixing nodules on soybean roots in a long-term field experiment. By means of various sequencing and genomic fingerprinting techniques, the repeated exposure to a mixture of tylosin, sulfamethazine, and chlortetracycline each at a nominal soil concentration of 0.1 mg · kg−1 soil was found to modify the diversity and identity of bradyrhizobia occupying the nodules. Nodule occupancy was not associated with the level of sensitivity to the antibiotics, indicating that the observed effects were not due to the direct toxicity of the antibiotics on bradyrhizobia. Altogether, these results indicate the potential for long-term impacts of antibiotics on this agronomically important symbiosis.