Characterization of Sclerotium rolfsii causing foot rot: a severe threat of betel vine cultivation in Bangladesh.

The development of the foot rot disease caused by the fungus Sclerotium rolfsii is one of the primary variables endangering betel vine production in Bangladesh. Consequently, with the ultimate objective of finding efficient preventive and control strategies for this infamous phytopathogen, the current study was undertaken for comprehensive population structure analysis, exploration of physiological features and incidence patterns of pathogenic S. rolfsii isolates. We discovered 22 S. rolfsii isolates from nine northern districts of Bangladesh. Mohanpur (51.90%), Bagmara (54.09%), and Durgapur (49.45%) upazilas in the Rajshahi district had the more severe occurrences of foot rot disease, while Chapainawabganj (18.89%) had the least number of cases. The isolates differed substantially in terms of morphology and growth rate. By employing the UPGMA algorithm to analyze the combined morphological data from 22 S. rolfsii isolates, these isolates were divided into six different groups with a 62% similarity level. Somatic incompatibility was also found in some isolates. The RAPD-4 primer confirmed 100% polymorphism among these isolates, and these genetic variations were further validated by molecular analysis. The results of the morphological and molecular analysis revealed that there was significant variation among the S. rolfsii isolates. Finally, a comprehensive characterization of S. rolfsii would allow for a suitable management strategy for betel vine's deadly foot rot disease. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03890-8.

Protocol for live imaging of intracellular nanoscale structures using atomic force microscopy with nanoneedle probes.

Atomic force microscopy (AFM) is capable of nanoscale imaging but has so far only been used on cell surfaces when applied to a living cell. Here, we describe a step-by-step protocol for nanoendoscopy-AFM, which enables the imaging of nanoscale structures inside living cells. The protocol consists of cell staining, fabrication of the nanoneedle probes, observation inside living cells using 2D and 3D nanoendoscopy-AFM, and visualization of the 3D data. For complete details on the use and execution of this protocol, please refer to Penedo et al. (2021)1 and Penedo et al. (2021).2.

Visualizing intracellular nanostructures of living cells by nanoendoscopy-AFM.

Atomic force microscopy (AFM) is the only technique that allows label-free imaging of nanoscale biomolecular dynamics, playing a crucial role in solving biological questions that cannot be addressed by other major bioimaging tools (fluorescence or electron microscopy). However, such imaging is possible only for systems either extracted from cells or reconstructed on solid substrates. Thus, nanodynamics inside living cells largely remain inaccessible with the current nanoimaging techniques. Here, we overcome this limitation by nanoendoscopy-AFM, where a needle-like nanoprobe is inserted into a living cell, presenting actin fiber three-dimensional (3D) maps, and 2D nanodynamics of the membrane inner scaffold, resulting in undetectable changes in cell viability. Unlike previous AFM methods, the nanoprobe directly accesses the target intracellular components, exploiting all the AFM capabilities, such as high-resolution imaging, nanomechanical mapping, and molecular recognition. These features should greatly expand the range of intracellular structures observable in living cells.

Cell penetration efficiency analysis of different atomic force microscopy nanoneedles into living cells.

Over the last decade, nanoneedle-based systems have demonstrated to be extremely useful in cell biology. They can be used as nanotools for drug delivery, biosensing or biomolecular recognition inside cells; or they can be employed to select and sort in parallel a large number of living cells. When using these nanoprobes, the most important requirement is to minimize the cell damage, reducing the forces and indentation lengths needed to penetrate the cell membrane. This is normally achieved by reducing the diameter of the nanoneedles. However, several studies have shown that nanoneedles with a flat tip display lower penetration forces and indentation lengths. In this work, we have tested different nanoneedle shapes and diameters to reduce the force and the indentation length needed to penetrate the cell membrane, demonstrating that ultra-thin and sharp nanoprobes can further reduce them, consequently minimizing the cell damage.