Abstract
Artificial surfaces are commonly used in place of leaves in phyllosphere microbiology to study microbial behaviour on plant leaf surfaces. These surfaces enable a reductionist approach to be undertaken, to enable individual environmental factors influencing microorganisms to be studied. Commonly used artificial surfaces include nutrient agar, isolated leaf cuticles, and reconstituted leaf waxes. Recently, replica surfaces mimicking the complex topography of leaf surfaces for phyllosphere microbiology studies are appearing in literature. Replica leaf surfaces have been produced in agar, epoxy, polystyrene, and polydimethylsiloxane (PDMS). However, none of these protocols are suitable for replicating fragile leaves such as of the model plant Arabidopsis thaliana. This is of importance, as A. thaliana is a model system for molecular plant genetics, molecular plant biology, and microbial ecology. To overcome this limitation, we introduce a versatile replication protocol for replicating fragile leaf surfaces into PDMS. Here we demonstrate the capacity of our replication process using optical microscopy, atomic force microscopy (AFM), and contact angle measurements to compare living and PDMS replica A. thaliana leaf surfaces. To highlight the use of our replica leaf surfaces for phyllosphere microbiology, we visualise bacteria on the replica leaf surfaces in comparison to living leaf surfaces.
Highlights
In phyllosphere microbiology, the study of microorganisms that reside on the leaves of plants, artificial surfaces are used to provide an insight into microbial behaviour in the phyllosphere[14]
Our results revealed that PDMS is the most suitable replica leaf replica material, due to the high fidelity and favourable degradation characteristics achieved with PDMS, while exhibiting comparable hydrophobicity and bacterial survival characteristics observed on generic isolated leaf cuitcles[20]
Growing plants in optimal conditions in either soil or culture media does not influence the topography of the leaf surface, our protocol needed to work for both soil and culture media grown plants to enable microbiologists to replica their own plant leaves for their own phyllosphere studies
Summary
The study of microorganisms that reside on the leaves of plants, artificial surfaces are used to provide an insight into microbial behaviour in the phyllosphere[14]. Artificial surfaces include: (1) flat surfaces, such as nutrient agar, and inert surfaces (i.e. stainless steel); and (2) microstructured surfaces, such as isolated leaf cuticles, leaf peels, reconstituted leaf waxes, and microfabricated surfaces[13,15,16,17,18,19,20] These surfaces are suitable for their intended purpose, they do not fully represent the complex topography of plant leaves required for some phyllosphere microbiology studies[21,22]. This obvious shortcoming has led to the development of protocols utilizing double-casting approaches to produce leaf replicas in agar, epoxy, polystyrene, and polydimethylsiloxane (PDMS)[22,23,24,25,26]. Light curable polymers produce undesirable chemical and ultraviolet (UV) light exposure to the plant leaf[41]
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