Aptamer-based technology for detecting Bacillus subtilis in soil.

The uncertainty associated with the impact of a bioinoculant on soil microbial community and, as a consequence, on soil quality, as well as the need to define its persistence, has prompted the demand for an accurate detection and tracking of the presence and the quantification of a target microbial inoculant in soil. Although DNA or RNA-based molecular detection are well established and commonly applied in this regard, alternative ligands such as DNA-aptamers have several advantages over them, such as low cost, ease of modification, ease of immobilisation on lab-on-chip or nanosensors, high stability and not thermolability. In this study, we used a toggle-cell SELEX method to isolate, select and characterise ssDNA (single-strand DNA) aptamers to detect a Bacillus subtilis strain which is being tested as a plant growth promoting rhizobacteria (PGPR) formulation. Two ssDNA aptamers (patenting application n.102022000022590) showed strong affinity and specificity for B. subtilis strains, with values of the kinetic parameters Kd (dissociation constant) in the nanomolar range and Bmax (maximum intensity of binding) around 1. Validation of the suitability of the aptamers was validated on three inoculated soils characterised by different chemical-physical features and in soil from a field trial with the formulated B. subtilis PCM/B 00105 strain. These are considered significant features to monitor B. subtilis strains in soil, practical to optimise bioinoculant application methods, support regulatory processes and foster the shift of agricultural production toward more sustainable cropping systems. KEY POINTS: • First DNA aptamers binding a B. subtilis strain included in a bioinoculum formulation. • First DNA aptamer binding B. subtilis in soil. • Aptamer may be a method for microbial inoculant detection in soil.

Quantification of nitrogen cycle functional genes from viable archaea and bacteria in paddy soil.

AIMS: One of the main challenges of culture-independent soil microbiology is distinguishing the microbial community's viable fraction from dead matter. Propidium monoazide (PMA) binds the DNA of dead cells, preventing its amplification. This dye could represent a robust means to overcome the drawbacks of other selective methods, such as ribonucleic acid-based analyses. METHODS AND RESULTS: We quantified functional genes from viable archaea and bacteria in soil by combining the use of PMA and quantitative polymerase chain reaction. Four N-cycle-related functional genes (bacterial and archaeal ammonia monooxygenase, nitrate reductase, and nitrite reductase) were successfully quantified from the living fraction of bacteria and archaea of a paddy soil. The protocol was also tested with pure bacterial cultures and soils with different physical and chemical properties. CONCLUSIONS: The experiment results revealed a contrasting impact of mineral and organic fertilizers on the abundance of microbial genes related to the N-cycle in paddy soil.

The impact of Beauveria species bioinocula on the soil microbial community structure in organic strawberry plantations.

Introduction: The multifunctionality of microorganisms, including entomopathogenic fungi, represents a feature that could be exploited to support the development, marketing, and application of microbial-based products for plant protection. However, it is likely that this feature could affect the composition and dynamics of the resident soil microorganisms, possibly over a longer period. Therefore, the methodology utilized to evaluate such impact is critical for a reliable assessment. The present study was performed to evaluate the impact of strains of Beauveria brongniartii and Beauveria bassiana on soil bacterial and fungal communities using an approach based on the terminal restriction fragment polymorphism (T-RFLP) analysis. Materials and methods: Soil samples in the vicinity of the root system were collected during a 3-year period, before and after the bioinocula application, in two organic strawberry plantations. Specific primers were used for the amplification of the bacterial 16S rRNA gene and the fungal ITS region of the ribosome. Results and discussion: Data of the profile analysis from T-RFLP analysis were used to compare the operational taxonomic unit (OTU) occurrence and intensity in the inoculated soil with the uninoculated control. With regard to the impact on the bacterial community, both Beauveria species were not fully consistently affecting their composition across the seasons and fields tested. Nevertheless, some common patterns were pointed out in each field and, sometimes, also among them when considering the time elapsed from the bioinoculum application. The impact was even more inconsistent when analyzing the fungal community. It is thus concluded that the application of the bioinocula induced only a transient and limited effect on the soil microbial community, even though some changes in the structure dynamic and frequency of soil bacterial and fungal OTUs emerged.

Current Methods, Common Practices, and Perspectives in Tracking and Monitoring Bioinoculants in Soil.

Microorganisms promised to lead the bio-based revolution for a more sustainable agriculture. Beneficial microorganisms could be a valid alternative to the use of chemical fertilizers or pesticides. However, the increasing use of microbial inoculants is also raising several questions about their efficacy and their effects on the autochthonous soil microorganisms. There are two major issues on the application of bioinoculants to soil: (i) their detection in soil, and the analysis of their persistence and fate; (ii) the monitoring of the impact of the introduced bioinoculant on native soil microbial communities. This review explores the strategies and methods that can be applied to the detection of microbial inoculants and to soil monitoring. The discussion includes a comprehensive critical assessment of the available tools, based on morpho-phenological, molecular, and microscopic analyses. The prospects for future development of protocols for regulatory or commercial purposes are also discussed, underlining the need for a multi-method (polyphasic) approach to ensure the necessary level of discrimination required to track and monitor bioinoculants in soil.

When Salt Meddles Between Plant, Soil, and Microorganisms.

In extreme environments, the relationships between species are often exclusive and based on complex mechanisms. This review aims to give an overview of the microbial ecology of saline soils, but in particular of what is known about the interaction between plants and their soil microbiome, and the mechanisms linked to higher resistance of some plants to harsh saline soil conditions. Agricultural soils affected by salinity is a matter of concern in many countries. Soil salinization is caused by readily soluble salts containing anions like chloride, sulphate and nitrate, as well as sodium and potassium cations. Salinity harms plants because it affects their photosynthesis, respiration, distribution of assimilates and causes wilting, drying, and death of entire organs. Despite these life-unfavorable conditions, saline soils are unique ecological niches inhabited by extremophilic microorganisms that have specific adaptation strategies. Important traits related to the resistance to salinity are also associated with the rhizosphere-microbiota and the endophytic compartments of plants. For some years now, there have been studies dedicated to the isolation and characterization of species of plants' endophytes living in extreme environments. The metabolic and biotechnological potential of some of these microorganisms is promising. However, the selection of microorganisms capable of living in association with host plants and promoting their survival under stressful conditions is only just beginning. Understanding the mechanisms of these processes and the specificity of such interactions will allow us to focus our efforts on species that can potentially be used as beneficial bioinoculants for crops.

Francisella tularensis: No Evidence for Transovarial Transmission in the Tularemia Tick Vectors Dermacentor reticulatus and Ixodes ricinus.

BACKGROUND: Tularemia is a zoonosis caused by the Francisella tularensis, a highly infectious Gram-negative coccobacillus. Due to easy dissemination, multiple routes of infection, high environmental contamination and morbidity and mortality rates, Francisella is considered a potential bioterrorism threat and classified as a category A select agent by the CDC. Tick bites are among the most prevalent modes of transmission, and ticks have been indicated as a possible reservoir, although their reservoir competence has yet to be defined. Tick-borne transmission of F. tularensis was recognized in 1923, and transstadial transmission has been demonstrated in several tick species. Studies on transovarial transmission, however, have reported conflicting results. OBJECTIVE: The aim of this study was to evaluate the role of ticks as reservoirs for Francisella, assessing the transovarial transmission of F. tularensis subsp. holarctica in ticks, using experimentally-infected females of Dermacentor reticulatus and Ixodes ricinus. RESULTS: Transmission electron microscopy and fluorescence in situ hybridization showed F. tularensis within oocytes. However, cultures and bioassays of eggs and larvae were negative; in addition, microscopy techniques revealed bacterial degeneration/death in the oocytes. CONCLUSIONS: These results suggest that bacterial death might occur in oocytes, preventing the transovarial transmission of Francisella. We can speculate that Francisella does not have a defined reservoir, but that rather various biological niches (e.g. ticks, rodents), that allow the bacterium to persist in the environment. Our results, suggesting that ticks are not competent for the bacterium vertical transmission, are congruent with this view.

Solid phase treatment of an aged soil contaminated by polycyclic aromatic hydrocarbons.

Laboratory scale tests were carried out in order to evaluate the removal efficiency of polyaromatic hydrocarbons (PAHs) during the different biological treatments of a Manufacturing Gas Plant site aged soil, heavily contaminated by high molecular weight compounds. Biodegradation studies were carried out at nearly 25 degrees C in solid phase reactors. Three tests were performed, over a period of 100 days for each test. In the first test (P1-bioaugmentated), soil was mixed with wood chips and urea at the start of the treatment and after six weeks from the beginning of the test was also periodically inoculated (at 42, 54, 69, 82, and 96 days) with selected consortia of autochthonous PAH-degrading bacteria. The second test (P2-biostimulated) was performed similarly to the previous one, but without any inoculations. In the third test (P3-control) only soil was introduced. All systems were aerated daily and humidified at the occurrence. PAH concentration, total cultivable heterotrophs, PAH-degrading bacteria, mycetes, pH, ATP concentration, and enzymatic activities were monitored every two weeks during the treatments. Tests showed that nearly 50% of light (three rings) PAHs, 35% of benzo-PAHs and 40% of the total PAHs could be removed in the reactor P2 following 100 days of treatment. Lower removal efficiency could be observed for light PAHs (28%) in the inoculated reactor (P1) at the end of the treatment: comparable abatements were obtained for benzo- and total PAHs. In the reactor P3 (control), the concentration of all polyaromatic hydrocarbons was nearly always constant, suggesting that the physical losses were negligible during the solid phase treatments. Therefore the C to N ratio balance resulted to be the key factor in promoting the biodegradation process of all PAHs.