Chip Technologies for Screening Chemical and Biological Agents Against Plant-Parasitic Nematodes

Abstract

Plant-parasitic nematodes cause substantial damage to agricultural crops worldwide. Long-term management of these pests requires novel strategies to reduce infection of host plants. Disruption of nematode chemotaxis to root systems has been proposed as a potential management approach, and novel assays are needed to test the chemotactic behavior of nematodes against a wide range of synthetic chemicals and root exudates. Two microfluidic chips were developed that measure the attraction or repulsion of nematodes to chemicals ("chemical chip") and young plant roots ("root chip"). The chip designs allowed for chemical concentration gradients to be maintained up to 24 h, the nematodes to remain physically separate from the chemical reservoirs, and for images of nematode populations to be captured using either a microscope or a flatbed scanner. In the experiments using the chemical chips, seven ionic solutions were tested on second-stage juveniles (J2s) of Meloidogyne incognita and Heterodera glycines. Results were consistent with previous reports of repellency of M. incognita to a majority of the ionic solutions, including NHNO, KNO, KCl, MgCl, and CaCl. H. glycines was found to be attracted to both NHNO and KNO, which has not been reported previously. A software program was written to aid in monitoring the location of nematodes at regular time intervals using the root chip. In experiments with the root chip, H. glycines J2s were attracted to roots of 3-day-old, susceptible (cultivar Williams 82) soybean seedlings, and attraction of H. glycines to susceptible soybean was similar across the length of the root. Attraction to resistant (cultivar Jack) soybean seedlings relative to the water only control was inconsistent across runs, and H. glycines J2s were not preferentially attracted to the roots of resistant or susceptible cultivars when both were placed on opposite sides of the same root chip. The chips developed allow for direct tests of plant-parasitic nematode chemotaxis to chemicals and roots with minimal human intervention.

Avicta and Clariva Affect the Biology of the Soybean Cyst Nematode, Heterodera glycines

“…So it is possible some treatment effects may have been missed due to lower movement of the J2s in our studies. Still, J2s in the control treatments in our experiments were significantly attracted to KNO 3 and significantly repelled from CaCl 2 just as reported by Beeman et al (2016), indicating that our assays were valid. It is likely that Avicta may not diffuse through the microfluidic filters into the lanes of the chemotaxis chips given the insolubility of Fig.…”

“…Active J2s (15-60) were added to the center nematode entry port of a microfluidic chemotaxis chip ( Supplementary Fig. S1) filled with sterile distilled water, as described by Beeman et al (2016). Soil leachate (30 ml), collected as described above, was added to a treatment port of a randomly assigned chip 1-14, lane (1-4), and side (left or right) combination.…”

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

“…The debris and egg-filled cysts (dead nematode females) were collected on the 250-μm-pore sieve and then transferred to a 3.7-cm-diameter, 250-μm-pore sieve and were crushed using a motorized rubber stopper to release the eggs [5]. These eggs (along with similar sized debris) were collected on a 15-cm-diameter sieve with 37-μm-diameter pores and then transferred into a microwavable container [11].…”

Lab-on-a-Chip Technologies for the Single Cell Level: Separation, Analysis, and Diagnostics

“…Some salient examples thereof are circulating tumor cells (CTC) of various types of cancer [1,[36][37][38], rare cells (e.g., sickle-cell variants of red blood cells) [39,40], parasites, like Plasmodium falciparum [1,7,10,11,[13][14][15][21][22][23]36,[41][42][43][44][45][46][47][48][49][50] and Trypanosoma spp. [8,44,[51][52][53][54][55][56][57] and even plant pathogens [26,58], as well as-after cells have been lysed-subcellular infection markers (e.g., DNA, RNA fragments) [10,11,22,43,[59][60][61][62]. Given the vast adaptability of microfluidics to any kind of single or multi-cellular assay [63], the ability to combine it with various light microscopy techniques…”

“…One very prominent aspect of microfluidic research is diagnostics on the single-cell or even molecular level. The significance of recent research towards microfluidics-based single cell diagnostic chips is apparent in health care, in our homes, and also very prominently in the fight against the COVID 19 pandemic: The diagnostic targets range from circulating tumor cells (CTC) [1][2][3][4][5][6], over parasites in blood [7][8][9][10][11][12][13][14][15], male fertility [16][17][18][19][20], molecular markers for infections [11,15,[21][22][23], cells of a specific stage in their life cycle [24,25], plant pathogens [26] and the SARS-CoV-2 proteome [27][28][29][30][31][32]. Depending on the exact target (either the entire cell or sub-cellular markers) there are different approaches to on-chip detection, each with their own underlying fundamentals and set of limitations.…”