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.

Microbial-Based Products to Control Soil-Borne Pathogens: Methods to Improve Efficacy and to Assess Impacts on Microbiome.

Microbial-based products (either as biopesticide or biofertilizers) have a long history of application, though their use is still limited, mainly due to a perceived low and inconsistent efficacy under field conditions. However, their efficacy has always been compared to chemical products, which have a completely different mechanism of action and production process, following the chemical paradigm of agricultural production. This paradigm has also been applied to regulatory processes, particularly for biopesticides, making the marketing of microbial-based formulations difficult. Increased knowledge about bioinocula behavior after application to the soil and their impact on soil microbiome should foster better exploitation of microbial-based products in a complex environment such as the soil. Moreover, the multifunctional capacity of microbial strains with regard to plant growth promotion and protection should also be considered in this respect. Therefore, the methods utilized for these studies are key to improving the knowledge and understanding of microbial-based product activity and improving their efficacy, which, from farmers' point of view, is the parameter to assess the usefulness of a treatment. In this review, we are thus addressing aspects related to the production and formulation process, highlighting the methods that can be used to evaluate the functioning and impact of microbial-based products on soil microbiome, as tools supporting their use and marketing.

Safety Level of Microorganism-Bearing Products Applied in Soil-Plant Systems.

The indiscriminate use of chemical fertilizers adversely affects ecological health and soil microbiota provoking loss of soil fertility and greater pathogen and pest presence in soil-plant systems, which further reduce the quality of food and human health. Therefore, the sustainability, circular economy, environmental safety of agricultural production, and health concerns made possible the practical realization of eco-friendly biotechnological approaches like organic matter amendments, biofertilizers, biopesticides, and reuse of agro-industrial wastes by applying novel and traditional methods and processes. However, the advancement in the field of Biotechnology/Agriculture is related to the safety of these microorganism-bearing products. While the existing regulations in this field are well-known and are applied in the preparation and application of waste organic matter and microbial inoculants, more attention should be paid to gene transfer, antibiotic resistance, contamination of the workers and environment in farms and biotech-plants, and microbiome changes. These risks should be carefully assessed, and new analytical tools and regulations should be applied to ensure safe and high-quality food and a healthy environment for people working in the field of bio-based soil amendments.

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.

Special Issue: Microorganisms and Plant Nutrition.

Plant-beneficial microorganisms affect plant nutrition and health, as a key part of prebiotic-, probiotic-, and symbiotic-based interactions [...].

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.

A Genomic and Transcriptomic Study on the DDT-Resistant Trichoderma hamatum FBL 587: First Genetic Data into Mycoremediation Strategies for DDT-Polluted Sites.

Trichoderma hamatum FBL 587 isolated from DDT-contaminated agricultural soils stands out as a remarkable strain with DDT-resistance and the ability to enhance DDT degradation process in soil. Here, whole genome sequencing and RNA-Seq studies for T. hamatum FBL 587 under exposure to DDT were performed. In the 38.9 Mb-genome of T. hamatum FBL 587, 10,944 protein-coding genes were predicted and annotated, including those of relevance to mycoremediation such as production of secondary metabolites and siderophores. The genome-scale transcriptional responses of T. hamatum FBL 587 to DDT exposure showed 1706 upregulated genes, some of which were putatively involved in the cellular translocation and degradation of DDT. With regards to DDT removal capacity, it was found upregulation of metabolizing enzymes such as P450s, and potentially of downstream DDT-transforming enzymes such as epoxide hydrolases, FAD-dependent monooxygenases, glycosyl- and glutathione-transferases. Based on transcriptional responses, the DDT degradation pathway could include transmembrane transporters of DDT, antioxidant enzymes for oxidative stress due to DDT exposure, as well as lipases and biosurfactants for the enhanced solubility of DDT. Our study provides the first genomic and transcriptomic data on T. hamatum FBL 587 under exposure to DDT, which are a base for a better understanding of mycoremediation strategies for DDT-polluted sites.

Fermentation Strategies to Improve Soil Bio-Inoculant Production and Quality.

The application of plant beneficial microorganisms has been widely accepted as an efficient alternative to chemical fertilizers and pesticides. Isolation and selection of efficient microorganisms, their characterization and testing in soil-plant systems are well studied. However, the production stage and formulation of the final products are not in the focus of the research, which affects the achievement of stable and consistent results in the field. Recent analysis of the field of plant beneficial microorganisms suggests a more integrated view on soil inoculants with a special emphasis on the inoculant production process, including fermentation, formulation, processes, and additives. This mini-review describes the different groups of fermentation processes and their characteristics, bearing in mind different factors, both nutritional and operational, which affect the biomass/spores yield and microbial metabolite activity. The characteristics of the final products of fermentation process optimization strategies determine further steps of development of the microbial inoculants. Submerged liquid and solid-state fermentation processes, fed-batch operations, immobilized cell systems, and production of arbuscular mycorrhiza are presented and their advantages and disadvantages are discussed. Recommendations for further development of the fermentation strategies for biofertilizer production are also considered.

Biocontrol of Melolontha spp. Grubs in Organic Strawberry Plantations by Entomopathogenic Fungi as Affected by Environmental and Metabolic Factors and the Interaction with Soil Microbial Biodiversity.

The efficacy of two strains of two Beauveria species (B. bassiana and B. brongniartii), individually or as co-inoculants, to control Melolontha sp. grubs was assessed in two organic strawberry plantations in relation to the environmental conditions, their abundance after soil inoculation, and their in vitro chitinolytic activity, thereby also verifying their impact on soil microbial communities. A reduction of the grubs' damage to strawberry plants was observed when compared to the untreated control in one plantation, irrespective of the strain used and whether they were applied as single or as co-inoculum. The metabolic pattern expressed by the two fungi in vitro was different: B. bassiana showed a higher metabolic versatility in the use of different carbon sources than B. brongniartii, whose profile was partly overlapped in the co-inoculum. Similar differences in the chitinolytic activity of each of the fungi and the co-inoculum were also pointed out. A higher abundance of B. bassiana in the soils receiving this species in comparison to those receiving B. brongniartii, together with its in vitro metabolic activity, could account for the observed diverse efficacy of pest damage control of the two species. However, environmental and climatic factors also affected the overall efficacy of the two bioinocula. According to the monitoring of the two species in soil, B. bassiana could be considered as a common native species in the studied locations in contrast to B. brongniartii, which seemed to be a non-endemic species. Nevertheless, the inoculation with both species or the co-inoculum did not consistently affect the soil microbial (fungi and bacteria) biodiversity, as expressed by the operational taxonomic unit (OTU) number and Shannon-Wiener diversity index based on terminal restriction fragment length polymorphism (TRFLP) data. A small transient increase of the share of the inoculated species to the total fungal community was noted by the analysis of genes copy numbers only for B. brongniartii at the end of the third growing season.

Aspegillus terreus: From Soil to Industry and Back.

Aspergillus terreus is an important saprophytic filamentous fungus that can be found in soils. Like many other soil microorganisms, A. terreus demonstrates multiple functions and offers various important metabolites, which can be used in different fields of human activity. The first application of A. terreus on an industrial level is the production of itaconic acid, which is now considered as one of the most important bioproducts in the Green Chemistry field. The general schemes for itaconic acid production have been studied, but in this mini-review some lines of future research are presented based on analysis of the published results. A. terreus is also intensively studied for its biocontrol activity and plant growth-promoting effect. However, this microorganism is also known to infect important crops such as, amongst others, rice, wheat, potato, sugar cane, maize, and soybean. It was suggested, however, that the balance between positive vs. negative effects is dependent on the soil-plant-inoculant dose system. A. terreus has frequently been described as an important human pathogen. Therefore, its safety manipulation in biotechnological processes for the production of itaconic acid and some drugs and its use in soil-plant systems should be carefully assessed. Some suggestions in this direction are discussed, particularly concerning the uses in crop production.

Monitoring of DDT in Agricultural Soils under Organic Farming in Poland and the Risk of Crop Contamination.

The analysis of 142 agricultural soil samples collected in organic farms across Poland with the intent to evaluate the level of DDT contamination resulted in more than 80% of the soils containing DDT. The ΣDDT (sum of all metabolites and isomers) concentration ranged between 0.005 and 0.383 mg/kg ΣDDT, with an average value of 0.064 mg/kg ΣDDT. However, the majority of plant samples collected from the crops growing on the sampled soils did not contain detectable DDT residues. The high DDT pollution levels detected in samples from four voivodeships (regions) among those monitored have been hypothesised to be linked to horticultural productions occurring to the sampled fields and typical of those regions, particularly in big-sized farms, during the period of DDT application, as well as the number of pesticides landfills present in these voivodeships. The elaboration of the o,p'-DDT/p,p'-DDT and DDT/(DDE + DDD) ratios to appraise the source or the period of contamination suggested that the contamination originated from past use of DDT rather than from impurities of more recent applications of other formulated substances. Such outcome thus suggests that the risk of contamination of organic products is likely derived from general environmental pollution levels rather than from the use of unauthorised substances in organic farming productions. Data from a trial with artificial contamination of soils indicated that using the DDT/(DDE + DDD) ratio in the presence of a low level of contamination could be less reliable than in highly contaminated soils.

Field Exploitation of Multiple Functions of Beneficial Microorganisms for Plant Nutrition and Protection: Real Possibility or Just a Hope?

Bioproducts, i.e., microbial based pesticides or fertilizers (biopesticides and biofertilizers), should be expected to play an ever-increasing role and application in agricultural practices world-wide in the effort to implement policies concerned with sustainable agriculture. However, several microbial strains have proven the capacity to augment plant productivity by enhancing crop nutrition and functioning as biopesticides, or vice-versa. This multifunctionality is an issue that is still not included as a concept and possibility in any legal provision regarding the placing on the market of bioproducts, and indicates difficulties in clearly classifying the purpose of their suitability. In this review, we overview the current understanding of the mechanisms in plant-microbe interactions underlining the dual function of microbial strains toward plant nutrition and protection. The prospects of market development for multifunctional bioproducts are then considered in view of the current regulatory approach in the European Union, in an effort that wants to stimulate a wider adoption of the new knowledge on the role played by microorganisms in crop production.

Formulation of Microbial Inoculants by Encapsulation in Natural Polysaccharides: Focus on Beneficial Properties of Carrier Additives and Derivatives.

In the last 10-15 years, the wide application of bioformulated plant beneficial microorganisms is accepted as an effective alternative of chemical agro-products. Two main problems can be distinguished in their production and application: (a) economical competiveness based on the overall up-stream and down-stream operational costs, and (b) development of commercial products with a high soil-plant colonization potential in controlled conditions but not able to effectively mobilize soil nutrients and/or combat plant pathogens in the field. To solve the above problems, microbe-based formulations produced by immobilization methods are gaining attention as they demonstrate a large number of advantages compared to other solid and liquid formulations. This mini-review summarizes the knowledge of additional compounds that form part of the bioformulations. The additives can exert economical, price-decreasing effects as bulking agents or direct effects improving microbial survival during storage and after introduction into soil with simultaneous beneficial effects on soil and plants. In some studies, combinations of additives are used with a complex impact, which improves the overall characteristics of the final products. Special attention is paid to polysaccharide carriers and their derivates, which play stimulatory role on plants but are less studied. The mini-review also focuses on the potential difficulty in evaluating the effects of complex bio-formulations.

Bioremediation of Dichlorodiphenyltrichloroethane (DDT)-Contaminated Agricultural Soils: Potential of Two Autochthonous Saprotrophic Fungal Strains.

DDT (dichlorodiphenyltrichloroethane) was used worldwide as an organochlorine insecticide to control agricultural pests and vectors of several insect-borne human diseases. It was banned in most industrialized countries; however, due to its persistence in the environment, DDT residues remain in environmental compartments, becoming long-term sources of exposure. To identify and select fungal species suitable for bioremediation of DDT-contaminated sites, soil samples were collected from DDT-contaminated agricultural soils in Poland, and 38 fungal taxa among 18 genera were isolated. Two of them, Trichoderma hamatum FBL 587 and Rhizopus arrhizus FBL 578, were tested for tolerance in the presence of 1-mg liter-1 DDT concentration by using two indices based on fungal growth rate and biomass production (the tolerance indices Rt:Rc and TI), showing a clear tolerance to DDT. The two selected strains were studied to evaluate catabolic versatility on 95 carbon sources with or without DDT by using the Phenotype MicroArray system and to investigate the induced oxidative stress responses. The two strains were able to use most of the substrates provided, resulting in both high metabolic versatility and ecological functionality in the use of carbon sources, despite the presence of DDT. The activation of specific metabolic responses with species-dependent antioxidant enzymes to cope with the induced chemical stress has been hypothesized, since the presence of DDT promoted a higher formation of reactive oxygen species in fungal cells than the controls. The tested fungi represent attractive potential candidates for bioremediation of DDT-contaminated soil and are worthy of further investigations.IMPORTANCE The spread and environmental accumulation of DDT over the years represent not only a threat to human health and ecological security but also a major challenge because of the complex chemical processes and technologies required for remediation. Saprotrophic fungi, isolated from contaminated sites, hold promise for their bioremediation potential toward toxic organic compounds, since they might provide an environment-friendly solution to contamination. Once we verified the high tolerance of autochthonous fungal strains to high concentrations of DDT, we showed how fungi from different phyla demonstrate a high metabolic versatility in the presence of DDT. The isolates showed the singular ability to keep their functionality, despite the DDT-induced production of reactive oxygen species.

Potential application of glycerol in the production of plant beneficial microorganisms.

This review highlights the importance of research for development of biofertilizer and biocontrol products based on the use of glycerol for further process scale-up to industrial microbiology. Glycerol can be used successfully in all stages of production of plant beneficial microorganisms. It serves as an excellent substrate in both submerged and solid-state fermentation processes with free and immobilized microbial cells. Glycerol is also one of the most attractive formulation agents that ensures high cell density and viability including in harsh environmental conditions. Future research is discussed to make this inexpensive material a base for industrial production of plant beneficial microorganisms.

Antagonistic potential of Pseudomonas graminis 49M against Erwinia amylovora, the causal agent of fire blight.

In a previous study (Mikicinski et al. in Eur J Plant Pathol, doi: 10.1007/s10658-015-0837-y , 2015), we described the characterization of novel strain 49M of Pseudomonas graminis, isolated from the phyllosphere of apple trees in Poland showing a good protective activity against fire blight on different organs of host plants. We now report investigations to clarify the basis for this activity. Strain 49M was found to produce siderophores on a medium containing complex CAS-Fe(3+) and HDTMA, but was not able to produce N-acyl homoserine lactones (AHLs). Moreover, it formed a biofilm on polystyrene and polyvinyl chloride (PVC) surfaces. Strain 49M gave a positive reaction in PCR with primers complementary to gacA, the regulatory gene influencing the production of several secondary metabolites including antibiotics. The genes prnD (encoding pyrrolnitrin), pltC, pltB (pyoluteorin), phlD (2,4-diacetyl-phloroglucinol) and phzC as well as phzD (and their homologs phzF and phzA encoding phenazine), described for antagonistic fluorescent pseudomonads, however, were not detected. Research into the biotic relationship between strain 49M and Erwinia amylovora strain Ea659 on five microbiological media showed that this strain clearly inhibited the growth of the pathogen on King's B and nutrient agar with glycerol media, to a very small extent on nutrient agar with sucrose, and not at all on Luria-Bertani agar. On medium 925, strain 49M even stimulated E. amylovora growth. The addition of ferric chloride to King's B resulted in the loss of its inhibitory ability. Testing the survival of 49M in vitro showed its resistance to drought, greater than that of E. amylovora.