New methods of removing debris and high-throughput counting of cyst nematode eggs extracted from field soil

Autoři: Upender Kalwa aff001;  Christopher Legner aff001;  Elizabeth Wlezien aff002;  Gregory Tylka aff002;  Santosh Pandey aff001
Působiště autorů: Department of Electrical and Computer Engineering, Iowa State University, Ames, Iowa, United States of America aff001;  Department of Plant Pathology and Microbiology, Iowa State University, Ames, Iowa, United States of America aff002
Vyšlo v časopise: PLoS ONE 14(10)
Kategorie: Research Article


The soybean cyst nematode (SCN), Heterodera glycines, is the most damaging pathogen of soybeans in the United States. To assess the severity of nematode infestations in the field, SCN egg population densities are determined. Cysts (dead females) of the nematode must be extracted from soil samples and then ground to extract the eggs within. Sucrose centrifugation commonly is used to separate debris from suspensions of extracted nematode eggs. We present a method using OptiPrep as a density gradient medium with improved separation and recovery of extracted eggs compared to the sucrose centrifugation technique. Also, computerized methods were developed to automate the identification and counting of nematode eggs from the processed samples. In one approach, a high-resolution scanner was used to take static images of extracted eggs and debris on filter papers, and a deep learning network was trained to identify and count the eggs among the debris. In the second approach, a lensless imaging setup was developed using off-the-shelf components, and the processed egg samples were passed through a microfluidic flow chip made from double-sided adhesive tape. Holographic videos were recorded of the passing eggs and debris, and the videos were reconstructed and processed by custom software program to obtain egg counts. The performance of the software programs for egg counting was characterized with SCN-infested soil collected from two farms, and the results using these methods were compared with those obtained through manual counting.

Klíčová slova:

Computer software – Deep learning – Filter paper – Imaging techniques – Microfluidics – Sucrose – Centrifugation – Density gradient centrifugation


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2019 Číslo 10
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