Loop-mediated isothermal amplification (LAMP) assay coupled with gold nanoparticles for colorimetric detection of Trichoderma spp. in Agaricus bisporus cultivation substrates

One of the significant challenges in organic cultivation of edible mushrooms is the control of invasive. Trichoderma species that can hinder the mushroom production and lead to economic losses. Here, we present a novel loop-mediated isothermal amplification (LAMP) assay coupled with gold nanoparticles (AuNPs) for rapid colorimetric detection of Trichoderma spp. The specificity of LAMP primers designed on the tef1 gene was validated in silico and through gel-electrophoresis on Trichoderma harzianum and non-target soil-borne fungal and bacterial strains. LAMP amplification of genomic DNA templates was performed at 65 °C for only 30 min. The results were rapidly visualized in a microplate format within less than 5 min. The assay is based on salt-induced aggregation of AuNPs that is being prevented by the amplicons produced in case of positive LAMP reaction. As the solution color changes from red to violet upon nanoparticle aggregation can be observed with the naked eye, the developed LAMP-AuNPs assay can be easily operated to provide a simple initial screening for the rapid detection of Trichoderma in button mushroom cultivation substrate.Cultivated edible mushrooms are a substantial nutritional source that holds economic significance in various regions across the world 1,2 . Asian countries are the largest mushroom producers contributing up to 76% of the global production (China alone produces about 35%), followed by Europe (17.2%) and United States (5.9%) 3 . The global market value of fresh mushrooms was estimated to US$ 38 billion in 2018 , and is expected to increase in future due to the changes in population lifestyle and growing consumers' food awareness. Mushrooms embody a rich source of healthy nutrients and are a staple in the human diet. They are characterized by a low-calorie content, are devoid of saturated fat and cholesterol, and encompass all essential amino acids. Apart from their nutritional value, the appeal of mushrooms lies in antioxidant activity and therapeutic properties. Additionally, their distinctive taste and unique texture make them an attractive choice for inclusion as a food ingredient or as a substitute for food additives 5-7 , both in traditional food products and in the rising field of alternative proteins. Modern mushroom farming elicits a number of environmental impacts which can be moderated through the circular economy principles based on upcycling of organic waste in the cultivation process. Button mushrooms (Agaricus bi