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Detection of biocontrol agents from casing material to inhibit mushroom mycoparasites through casing microbiome characterization and Raman spectroscopy of isotopically labelled organisms.

Periodic Reporting for period 1 - MYCOBIOME (Detection of biocontrol agents from casing material to inhibit mushroom mycoparasites through casing microbiome characterization and Raman spectroscopy of isotopically labelled organisms.)

Reporting period: 2017-03-13 to 2019-03-12

Edible mushrooms are a very healthy alternative for western diets. The commercial cultivation of mushrooms consists on the production of nutritional substrates for the development and fructification of selected mycelium. Bacteria from different genera play a significant role on mushroom cultivation. We have been working actively with the industry to evaluate the role of bacteria and fungi when interacting with the host to develop bio-solutions (H2020-MSCA-IF-2016/H2020-MSCA-IF-2016 - Grant Agreement number: 742966 - MYCOBIOME).
The project is a first research approach to design biosolutions to deal with mycoparasites. Currently the industry is using pesticides, therefore providing an alternative we aim to implement a greening of the industry, and, at the same time, to facilitate tools for the growers in order to produce healthier mushrooms for concern customers.
The overall objectives of the project were:
1) Selection of casing materials: agronomic behaviour and potential suppressive effect against mycoparasites.
2) Analysis of the casing-microbiome evolution along the crop cycle.
3) Isolation and incubation of target organisms.
4) Detection and isolation of bacteria showing selective antagonism towards L. fungicola and M. perniciosa.
5) Design of a synthetic microbial community to amend casing material. Biocontrol pilot trial in crop.
1) In this work three different casing materials were compared for their capacity to show a natural suppressive response against dry bubble and wet bubble. The highest mushroom production was collected from crops cultivated using the mixed casing material and black peat, which were not significantly different in yield. However, artificial infection with mycoparasites resulted in similar yield losses irrespective of the casing material used, indicating that the casing materials do not confer advantages in disease suppression.
2) The composition of the microbiome of the 50:50 casing mixture along the crop cycle, compost and basidiomes, was evaluated through next generation sequencing (NGS) of the V3-V4 region of the bacterial 16S rRNA gene, and the fungal, ITS2 region. The naturally established casing microbiota could help to suppress disease development when inoculum levels were low, but was not effective in suppressing a high pressure of artificially introduced fungal inoculum. Determination of the composition of the casing microbiome paves the way for the development of synthetic casing communities that can be used to investigate the role of specific components of the casing microbiota in mushroom production and disease control.
3) Elaboration of a biobank with bacterial strains isolated from the the casing materials evaluated: three different casing materials, blonde peat, black peat and a 50:50 mixture of both materials. The biobank has been set up through initial major isolation of native cultivable microbiota using a set of different growing media in vitro. The strains were further isolated as individual colonies while taken individual colonies identified according to their phenotype.
4) Antifungal activity was detected from tree individuals from the biobank both against Lecanicillium fungicola and Mycogone perniciosa: strains CM5, CM19 and CM35. The strains show clear inhibition of mycoparasites when co-cultured in vitro. The selected strains have been molecularly identified (through Sanger sequencing, and through high-throughput sequencing of the draft genome) and the patent application is currently under review.
5) A commercial prototype of bacterial water-soluble powder has been designed for the used of the biocontrol agents discovered in crop. A first trial has been conducted in crop treating artificially infected crops. The biocontrol treatments did not show detrimental effects on Agaricus vegetative mycelium or production (which suggest selectivity), however none significant yield increase was detected during the trial.
"A good track of open access publications has been delivered:
Articles in peer-review journals:
1. Carrasco et al., 2019. Casing microbiome dynamics during button mushroom cultivation: implications for dry and wet bubble diseases. Microbiology. doi: 10.1099/mic.0.000792
2. Carrasco et al., 2018. Supplementation in mushroom crops and its impact on yield and quality. AMB Express, 8(1), 146. doi: 10.1186/s13568-018-0678-0
3. Carrasco et al., 2019. Growing edible mushrooms: a conversation between bacteria and fungi. (Under review).
Book chapters
1. Carrasco, J.; Tello, M. L.; Perez-Clavijo, M.; Preston, G. 2019. Biotechnological requirements for the commercial cultivation of macrofungi: substrate and casing layer. In: Biology of Macrofungi. Ed. Pratap Singh, B., Chakchhuak, L, Passari, K. Springer. ISBN 978-3-030-02622-6. 159-175 pp. doi: 10.1007/978-3030-02622-6_7.
2. Preston, G.; Carrasco, J.; Gea, F.G.; Navarro, M.J. 2019. Biological control of microbial pathogens in edible mushrooms. In: Biology of Macrofungi. Ed. Pratap-Singh, B., Chakchhuak, L, Passari, K. Springer. ISBN 978-3-030-02622-6. 305-317 pp. doi: 10.1007/978-3-030-02622-6_15.
Outreach publications:
1. Carrasco, J et al., 2019. Boletín de la Asociación Española de Cultivadores de Champiñón. 91, pp. 10 - 18. (http://ctich.com/images//pdf/Revistas-Asociacion/Revista-Asochamp-91.pdf).
2. Carrasco J. et al., 2018. Boletín de la Asociación Española de Cultivadores de Champiñón. 89, pp. 10 - 18. (https://www.ctich.com/images/pdf/Revistas-Asociacion/Revista_CTICH-89_.pdf).
3. Carrasco J., et al., 2017. Boletín de la Asociación Española de Cultivadores de Champiñón. 88, pp. 12 - 16. (https://www.ctich.com/images/pdf/Revistas-Asociacion/Revista_CTICH_88.pdf).
4. Carrasco J., et al., 2017. Boletín de la Asociación Española de Cultivadores de Champiñón. 87, pp. 22. (https://www.ctich.com/images/pdf/Revistas-Asociacion/RevistaCTICH87-min.pdf).
Work presented by the fellow in international conferences and workshops:
- Carrasco et al. Next Generation Sequencing (NGS) reveals the presence of fungal mycoparasites in button mushroom substrates: structure and dynamics of the native microbiome. 9th International Conference on Mushroom Biology and Mushroom Products (9ICMBMP). November 12-19, 2018. Shanghai and Zhangzhou, China. Poster.
- Carrasco et al., 2018. Casing microbiome reveals the absence of a natural suppressive response against bubble diseases in button mushroom crops. Focused Meeting 2018: Microbiomes Underpinning Agriculture. ""Microbiology Society"". Cork (Ireland). 1-2 October 2018. Oral.
Awards and distinctions
- 2018 Hongzhen Distinguished Presentation Award for outstanding research in Mushroom Biology and Mushroom Products at the 9ICMBMP Nov. 12-19, 2018. China. 10000CNY (1460$).
- BSPP Travel Fund award 2018 from the British Society of Plant Pathology. £1000. Travel award to Jaime Carrasco to attend the 9ICMBMP Nov. 12-19, 2018. China.
- BMS Small grant award 2018 from the British Mycological Society. £350. Travel award to Jaime Carrasco to attend the Focused Meeting 2018: Microbiomes Underpinning Agriculture. ""Microbiology Society"". Cork (Ireland). 1-2 October 2018.
The discovering patent of selected biocontrol agents is under review. The work continues post-grant in collaboration with the Partner Association with the aim of building a consortium to apply for the European call BBI-JU 2019.
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