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

Abstract

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.

Robotic agricultural instrument for automated extraction of nematode cysts and eggs from soil to improve integrated pest management

“…In this regard, we do not anticipate any major alterations to our system other than changing the sieves and time durations for some operations, as the wet-sieving and decanting extraction procedure is similar for the other biological particles mentioned. There are further opportunities to couple the extraction and counting steps where the processed sample from the robotic instrument is automatically transferred into high-throughput nematode egg counting platforms 24 .…”

“…plunger made of a rubber stopper and driven by an electric motor) is used to gently break open the cyst walls on the #60 sieve, thereby releasing the eggs which are collected on an even finer sieve (#500 with 25-µm-diameter pores) 22 . A nematode-counting slide or microfluidic flow chip is then used to view and count the nematode cysts or eggs 24 .…”

“…Firstly, manual handling is involved in multiple steps of soil processing (i.e. sieving, collecting, washing, grinding, and counting) that are laborious, time-consuming, and prone to inter-operator error and thus inconsistent analysis 24 . Secondly, soil diagnostics for cyst nematodes currently are done in specialized laboratories with trained personnel, and the time between submitting a sample to receiving the results can be more than a few weeks 25 , 26 .…”

Behavioral Monitoring Tool for Pig Farmers: Ear Tag Sensors, Machine Intelligence, and Technology Adoption Roadmap

“…One suggested scheme for processing the raw data from different electronic sensors is shown in Figure 6. The data processing starts by segmenting the incoming sensors' data into separate time windows, as described in our previous work on deep learning [24]. We compute a set of features that give a good spectral and temporal representation of the data (such as mean, standard deviation, energy, skewness, entropy, and auto-regressive coefficients).…”

Rapid Production of PDMS Microdevices for Electrodriven Separations and Microfluidics by 3D-Printed Scaffold Removal

“…Our setup does not need any highly expensive way of production, and it can be used for a few purposes. There are some other low-cost preparation methods, including laser cutting and scribing adhesive tape [31][32][33]. However, lasers are still more expensive.…”