Researchers from CREA (Italy) have developed an aptamer-based technology to track the fate of microbial inoculants used in agriculture.
Microbial-based products such as fertilisers (now named microbial biostimulants by EU legislation) and biopesticides may support plant nutrition and protection under abiotic and biotic stress conditions and are expected to play a key role in agricultural sustainability in the future. In the last decades, they have received considerable attention from researchers, manufacturers, and farmers, mainly because they might help to reduce the use of chemicals in agriculture, and their application is steadily increasing. Currently, the world market of products containing micro-organisms stands at around $10bn and $3bn for biopesticides and biostimulants, respectively.
However, the inoculation of the soil with such beneficial micro-organisms may affect its native microbial populations, with effects that depend on the soil’s chemical and physical characteristics and the environmental conditions (i.e., climate, agronomic practices, cropping systems, etc.). Furthermore, considering the pivotal role of soil microbial diversity for life-supporting functions, changes occurring to the soil microbial structure after applying microbial-based formulations may affect the overall soil health status with effects which can impact crop productivity, quality, and human health.
Thus, the field application of such products requires their registration at the EU and national levels, together with an indication by the manufacturer of various specifications and analytic methods, making it possible to trace their destiny in the environment and prove their medium- and long-term effectiveness. These aspects are closely connected with the ability of the micro-organism to adapt and persist in the (soil) environment. In this framework, which encompasses scientific, commercial, and regulatory aspects, the development of tools to monitor the introduced microbial species is of paramount importance, particularly to ensure a correct risk assessment in relation to the environment and human health.
The EXCALIBUR project
The EXCALIBUR project aims at deepening our understanding of the mechanisms underlying the soil microbiome changes composition and functioning upon bioinoculant application in horticulture, thus providing a soil biodiversity-driven management strategy for farmers. Innovative fermentation and formulation processes have also been carried out to optimise the efficacy of several novel multifunctional bioinoculants. New potentially commercial products were thus developed, and the results we got so far from the field trials showed that these products could support the common practices that are currently used in horticulture by achieving the same performance level but reducing chemical inputs. Most of the effort is now put into the assessment of soil biodiversity dynamics as well as plant-soil-microbe interactions. Several innovative actions are ongoing, such as the development of predicting models, the biodiversity-based Decision Support System (DSS), or molecular diagnostic kits for a quick but reliable assessment of soil health status. However, one of the project’s main goals was the development of a tool to detect the abundance and monitor the persistence (fate) of the bio-inocula that are applied to the soil, using DNA-based techniques for targeting species-specific gene sequences. Such a tool is considered essential for farmers, confirming successful inoculations and the persistence of bio-inocula in the soil, as well as for regulatory purposes.
Current detection approaches for microbial inoculants
Earlier detection approaches from culture-dependent tools, such as direct microscopic examination, plate profiling, and Fluorescent in Situ Hybridisation (FISH), have provided essential insights into the detection and localisation of target microbial inoculants in soil. These approaches led the way to culture-independent tools addressing the analysis of target microbial species of bioinocula and evaluating the bioinocula effect on microbial communities’ structure and diversity.
The rapid development of DNA and RNA-based analytical methods offered new opportunities to monitor microbial inoculants’ survival and interactions within a specific soil community. Indeed, a high degree of resolution is fundamental to evaluate the success or failure of bacteria or fungi inoculation, tracing the ‘introduced DNA’ in a mixture of genomes from thousands of different native organisms. Culture-independent methods have also effectively characterised the soil microbial assemblages in space and time, evaluating their functional and trophic interactions. Recent research in the ‘omic’ era has expanded our knowledge and understanding of microbial community assembly, but tracing the bioinocula in the soil is still not a straightforward task. Many methods have been developed to enable inventories of microbial species composition and a good understanding of dynamics and processes of biodiversity in bulk or rhizospheric soil, but generally are not suitable to follow the fate of a single species. For example, ‘DNA barcoding’ has been widely used across all life forms, including micro-organisms, to distinguish a species from another. The barcode is derived from a PCR amplicon of a target sequence used to identify (or barcode) a micro-organism distinguishing it from other species. However, DNA barcodes are not error-free, and single-species barcoding needs to be designed based on robust genetic distances to obtain unique and highly discriminant markers. The genetic variability of individual strains, sometimes closely related but different in genomic traits, is exploited to discriminate the individual species but may as well provide inaccurate identifications. Markers based on sequences characterised by amplified regions (SCAR) have been widely used as molecular probes for tracking the fate of fungi. SCAR markers are based on universal primers, i.e., sequences universally present with highly conserved flanking regions, which, however, can discriminate only at genera or species group levels but not at species level within a pool of micro-organisms. However, markers suitable to monitor or discriminate the introduced bioinocula from native soil strains should be species-specific.
In this context, the EFSA (European Food Safety Authority) is working to establish methods helpful in evaluating the risk and traceability of micro-organisms introduced into soil. The most significant difficulty in the research and development of molecular markers to be used in soil relies on the fact of being able to identify markers that are species- or strain-specific, i.e., which can discriminate a species or a strain thereof, from another one in the soil, which represents a heterogeneous and highly complex matrix. Indeed, billions of micro-organisms, many of which are unknown, reside in a gram of soil. Therefore, new strain-specific detection methods are needed.
An aptamer-based detection tool
In EXCALIBUR, researchers Loredana Canfora and Andrea Manfredini from CREA proposed an aptamer-based detection tool as these are successfully applied for clinical purposes and, only more recently, in monitoring food and heavy metal contamination. Still, they have never been used for agroindustry. Aptamers are emerging biosensors based on ssDNA or RNA capture probes that can bind various target ligands with high affinity and are cheaper and more sensitive than antibodies. An aptamer, advantageously enables recognition of the target strain at a cellular level without any need to extract nucleic acids, resulting in a considerable lowering of costs compared to the known methods, both in terms of man-hours and in terms of consumable materials. Furthermore, the use of an aptamer makes it possible to perform an innovative in situ analysis, never carried out in the field of soil microbial inoculants’ traceability.
The idea was thus to select at least one of them by ‘systematic evolution of ligands by exponential enrichment’ (SELEX) method for the diagnostic traceability of microbial-based inocula in soil. The method was developed and validated for the detection of the micro-organism Bacillus subtilis, a bacterial species widespread in soil having the potential function of plant growth stimulation or protection, and exploited in several formulations for agricultural applications. The complete genome of the target strain B. subtilis PCM/B00105 was sequenced to select the species-specific aptamers. Based on a bioinformatic analysis, a specific region of the genome was identified, on which a pair of primers was designed for the selection of discriminating aptamer candidates. The choice and selection of the gene region on which to design the pair of primers were decisive for the efficient and successful definition of unique aptamers (Italian patent n. 102022000022590). To validate the results obtained in vitro, an experiment with soils inoculated with a formulation containing the B. subtilis PCM/B 00105 strain was carried out. It was necessary to optimise the method of extracting the cells from soil samples, as impurities and interferences of soil compounds and soil texture can affect the extraction efficiency. The technique for detecting the micro-organism B. subtilis by means of employing an aptamer-based approach is advantageously transferrable onto a mobile device, for example, using a biosensor.
Lab-on-a-chip
As high-affinity ligands, aptamers can be chemically modified to increase their degree of affinity. This latter characteristic makes aptamers like antibodies, but they are more stable compared to the latter, do not induce immune responses, are capable of being immobilised on inert supports and are not thermolabile. Thus, aptamers can be easily transferred onto a nanoscale ‘lab-on-a-chip’ microfluidic system as opposed to other biomarkers/biosensors that are limited to being applied on a laboratory scale and entail higher costs and very long analytical times. In the EXCALIBUR project, we are trying to develop a biosensor chip-based (lab-on-a-chip, LoC) consisting of the aptamer immobilised and exploiting surface acoustic wave (SAW) technology.
Conclusions
Despite the challenges posed by the soil complex matrix, the successful implementation of modern methods for traceability and monitoring of microbial inoculants in soil is a crucial step towards a better understanding of ecological systems and the correct adoption of practices involving the use of microbial-based products. Knowledge of soil ecology will enable the widening of the opportunities derived from the use of microbial products and ultimately help us protect our environment. By adopting this advanced approach, we can promote environmentally friendly practices essential for preserving our planet’s delicate balance. Moreover, it may be successfully used to monitor any target organism, such as B. subtilis in soil, being practical to optimise bioinoculant application methods, support regulatory processes and foster the shift of agricultural production toward more sustainable cropping systems. In conclusion, using new methods for traceability and monitoring micro-organisms in soil is a vital investment in our future and will benefit future generations.
References
Malusà E, Berg G, Biere A, Bohr A, Canfora L, Jungblut AD, Kepka W, Kienzle J, Kusstatscher P, Masquelier S, Pugliese M, Razinger J, Tommasini MG, Vassilev N, Meyling NV, Xu X, Mocali S (2021). A holistic approach for enhancing the efficacy of soil microbial inoculants in agriculture: from lab to field scale. Glob J Agric Innov Res Dev, 8:176–190. https://doi.org/10.15377/2409-9813.2021.08.14
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Manfredini A, Malusà E, Costa C, Pallottino F, Mocali S, Pinzari F, Canfora L (2021) Current methods, common practices, and perspectives in tracking and monitoring bioinoculants in soil. Front Microbiol 12:698491. https://doi.org/10. 3389/fmicb.2021.698491
Manfredini, A., Malusà, E. & Canfora, L. Aptamer-based technology for detecting Bacillus subtilis in soil. Appl Microbiol Biotechnol 107, 6963–6972 (2023). https://doi.org/10.1007/s00253-023-12765-0
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Co-authors
Loredana Canfora, CREA-AA, loredana.canfora@crea.gov.it
Andrea Manfredini, CREA-AA, andrea.manfredini@crea.gov.it
Eligio Malusà, INHORT, eligio.malusa@inhort.pl & CREA-VE, eligio.malusa@crea.gov.it
Please note, this article will also appear in the seventeenth edition of our quarterly publication.
