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Effects of a fungal invasion on soil bacteria Flavia Pinzari; Anne D. Jungblut; Clark M.D.; Misra R.; Xu X.-M.; Chooneea D. Presentation by F.Pinzari at The World Congress of Soil Science 2022, which took place in Glasgow from 31st July - 5th August 2022 Pinzari F.1,2, Clark M.D.1, Misra R. 3, Chooneea D.3, Xu X.-M.4, Jungblut A.D.1 1Life Sciences Department, Natural History Museum, Cromwell Road, SW7 5BD London, UK 2Institute for Biological Systems, Council of National Research of Italy (CNR), Monterotondo (RM), Italy 3Core Research Laboratories, Molecular Biology, Natural History Museum, London, United Kingdom 4National Institute of Agricultural Botany, East Malling Research Station (EMR), East Malling, UK Fungal bioinoculants have a vast potential in agriculture because they can help increase crop yields and quality and reduce the application of chemicals. Their effectiveness has been widely tested (Malusà et al., 2016). However, little is known about the effect of bioinoculants on microbial assemblages in non-rhizospheric soil. A sudden artificial introduction of a fungal species in soil could theoretically impact the biodiversity of local microbial communities and lead to changes in nutrient availability (van Elsas et al., 2012). We assessed the impact of a competitive fungal inoculum, the globally-used biofertiliser Trichoderma afroharzianum T22, on soil microcosms to understand 1) to what extent the native microbial community richness and relative abundance are influenced by a fungal strain introduced to soil; 2) whether microbial taxa are resilient to the disturbance caused by the fungus; 3) how far the bioinoculant impacts the soil microorganisms functions. We used bacterial 16S rRNA gene amplicon sequencing (Illumina) and a shotgun metagenomic analysis (Oxford Nanopore Sequencing) to analyse the microbial communities in bioreactors after seven weeks of incubation with and without the fungus. The presence of the fungus had a negative impact on the abundance of some groups of bacteria, such as the genus Pseudomonas, and it stimulated the presence of species metabolically linked to the fungus, including chitin degrading Chitinophagaceae. In conclusion, the results suggest that more than an impact on bacteria's overall biodiversity, the fungus has favoured some groups at the expense of others, even creating new food webs and trophic niches. References Malusà E, Pinzari F, Canfora L (2016) Efficacy of Biofertilizers: Challenges to Improve Crop Production. In: D.P. Singh et al. (eds.), Microbial Inoculants in Sustainable Agricultural Productivity: Microbial Inoculants in Sustainable Agricultural Productivity, pp.17-40 Springer India doi.org/10.1007/978-81-322-2644-4_2 van Elsas JD, Chiurazzi M, Mallon CA, Elhottova D, Kristufek V, Salles JF. (2012) Microbial diversity determines the invasion of soil by a bacterial pathogen. Proc Natl Acad Sci U S A. 24;109(4):1159-64. doi: 10.1073/pnas.1109326109. Acknowledgements The NHM-affiliated author Flavia Pinzari was funded by the H2020-MSCA-IF-EF-SE project “AlienInSoil”. This project has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement No 892048., The NHM-affiliated author Flavia Pinzari was funded by the H2020-MSCA-IF-EF-SE project "AlienInSoil". This project has received funding from the European Union's Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement No 892048., {"references":["Malusà E, Pinzari F, Canfora L (2016) Efficacy of Biofertilizers: Challenges to Improve Crop Production. In: D.P. Singh et al. (eds.), Microbial Inoculants in Sustainable Agricultural Productivity: Microbial Inoculants in Sustainable Agricultural Productivity, pp.17-40 Springer India doi.org/10.1007/978-81-322-2644-4_2","van Elsas JD, Chiurazzi M, Mallon CA, Elhottova D, Kristufek V, Salles JF. (2012) Microbial diversity determines the invasion of soil by a bacterial pathogen. Proc Natl Acad Sci U S A. 24;109(4):1159-64. doi: 10.1073/pnas.1109326109."]}

Aims The increasingly widespread use of beneficial microbial inocula in agriculture gives rise to two primary needs: i) the assessment of the environmental risk, i.e. their impact on local soil microbiome and soil properties; ii) being able to track them and monitor their persistence and fate to both optimize their formulation and application method. In previous years, PCR-based methods have detected bacterial or fungal bioinoculant at the species or strain level. However, the selective detection, quantification, and monitoring of target microbial species in a complex ecosystem such as soil require that the tests possess high specificity and sensitivity. Methods and results The work proposes a quantitative real-time PCR detection method using TaqMan chemistry, showing high specificity and sensitivity for thePaenibacillus polymyxaK16 strain. The primer and probe sets were designed using the polymyxin gene cluster targetingpmxCandpmxEsequences. Validation tests showed that these assays allowed a discriminant and specific detection ofP. polymyxa K16in soil. Conclusion The TaqMan-assay developed could thus ensure the necessary level of discrimination required by commercial and regulatory purposes to detect and monitor the bioinoculant in soil.

A project (EXCALIBUR) has been started aiming to deepen the knowledge on soil biodiversity dynamics and its synergistic effects with prebiotic and probiotic approaches in horticulture. In this context, new multifunctional soil microbial inoculants (bio-inocula) and bio-effectors will be tested on three model crops of economic importance (tomato, apple, strawberry) under different experimental and open-field conditions across Europe. Excalibur plans to develop a comprehensive strategy of soil management improving the effectiveness of biocontrol and biofertilization practices in organic farming also through the development of a Decision Support System (DSS) and tools for monitoring inocula in the soil. Such activities are expected to encourage the adoption of best practices derived from the new strategy by the farmer’s increasing also the awareness on the potential benefits of soil biodiversity maintenance for crops’ performance.

Seed oxygen consumption has been correlated to seed vigour and quality (Bradford et al., 2013). The ability of oxygen to penetrate the tissues covering the embryo or the absorption of oxygen by the enclosing tissues was pointed out as the main mechanism capable of controlling the germination (Edelstein et Welbaum, 2011). ln the same way, carbon dioxide evolution by seeds at the initial phases of germination actually represents an overall indication of metabolism and activity. The imbibition of dry seeds begins the process of respiration that can have a very different performance depending on the plant species and the viability of the seed. A colorimetric test on seed respiration was developed since 1950 (Hempton and Tekrony, 1995) and is based on the reduction of colourless solution 2,3,5-tripheniltetrazolim chloride or bromide into insoluble 2,3,5-triphenilformazan red in colour. This solution acts as an indicator for detection of reduction processes that take place in living parts of the seed. We propose a new approach where the seed is not in contact with the dye, and the respiration of single seeds is measured in a multiwell system using a spectrophotometer according to the protocol by Campbell (2003). The new protocol can be easily applied also to the screening of the taxie or stimulant effects of matrices and products directly on the seeds, using respiration as a metabolic indicator.

An analysis of biodeterioration on different kind of materials.