The Effects of ILeVO and VOTiVO on Root Penetration and Behavior of the Soybean Cyst Nematode, Heterodera glycines.

The objective of this study was to determine the effects of ILeVO (fluopyram) and VOTiVO (Bacillus firmus I-1582) seed treatments on Heterodera glycines second-stage juvenile (J2) root penetration and behavior. In a growth chamber experiment, roots of soybeans grown from treated or untreated seeds were inoculated with H. glycines J2s at soil depths of 2.5, 5, or 7.5 cm. ILeVO significantly reduced H. glycines root penetration compared with the untreated control, but only when J2s were inoculated at a soil depth of 2.5 cm, which was near the seed. Changes in nematode behavior were assessed by collecting 60-s videos of J2s after 2 h of exposure to exudates from treated seeds or radicles from treated seeds or from soil leachates in which treated seeds were planted. X- and y-coordinates of each of the 13 reference points were recorded every hour for 24 h. A custom program analyzed and transformed the coordinates into nematode motion parameters (speed and total change in curvature). ILeVO, but not VOTiVO, seed exudates significantly reduced J2 speed relative to the untreated control. Soil leachates from ILeVO or VOTiVO treatments had no consistent effect on H. glycines speed or total change in curvature compared with the untreated control. In another experiment, treated or untreated seeds were incubated in wells of 6-well tissue culture plates containing 11.5% Pluronic gel. Seeds were removed after 2 h, and approximately 50 J2s then were pipetted into each well. The plates were scanned every 60 min for 24 h, and the number of J2s in each well that moved a minimum distance of ≥300 µm was determined using another custom software program. ILeVO, but not VOTiVO, significantly reduced the movement of J2 populations relative to control wells in which no seeds were added. And wells that had seeds, treated or not, yielded significantly less J2 movement compared with the no-seed control. The results of these experiments indicate that ILeVO reduces activity on H. glycines J2s but may not affect nematodes beyond a limited area surrounding the treated seed.

Movement and Motion of Soybean Cyst Nematode Heterodera glycines Populations and Individuals in Response to Abamectin.

Two new in vitro methods were developed to analyze plant-parasitic nematode behavior, at the population and the individual organism levels, through time-lapse image analysis. The first method employed a high-resolution flatbed scanner to monitor the movement of a population of nematodes over a 24-h period at 25°C. The second method tracked multiple motion parameters of individual nematodes on a microscopic scale, using a high-speed camera. Changes in movement and motion of second-stage juveniles (J2) of the soybean cyst nematode Heterodera glycines Ichinohe were measured after exposure to a serial dilution of abamectin (0.1 to 100 µg/ml). Movement and motion of H. glycines were significantly reduced as the concentration of abamectin increased. The effective range of abamectin to inhibit movement and motion of H. glycines J2 was between 1.0 and 10 µg/ml. Proof-of-concept experiments for both methods produced one of the first in vitro sensitivity studies of H. glycines to abamectin. The two methods developed allow for higher-throughput analysis of nematode movement and motion and provide objective and data-rich measurements that are difficult to achieve from conventional microscopic laboratory methods.

Assessing the Effects of ILeVO and VOTiVO Seed Treatments on Reproduction, Hatching, Motility, and Root Penetration of the Soybean Cyst Nematode, Heterodera glycines.

Successful management of the soybean cyst nematode Heterodera glycines is limited by increased virulence of nematode populations on resistant soybean cultivars and persistence of the nematode in the soil in the absence of hosts. Seed treatments are now available for H. glycines management. However, it is unclear how these treatments affect specific life stages of the nematode. The objectives of this study were to assess the effects of ILeVO (with active ingredient fluopyram) and VOTiVO (with active ingredient Bacillus firmus I-1582) seed treatments on H. glycines reproduction and important processes in the nematode life cycle, such as second-stage juvenile (J2) hatching, motility, and root penetration. The effects of seed treated with formulated (ILeVO and VOTiVO) and nonformulated active ingredient (fluopyram and B. firmus I-1582) on H. glycines reproduction were conducted in a greenhouse. Nematode reproduction on plants grown from seed treated with ILeVO or technical fluopyram (active ingredient only) was reduced by 35 to 97% relative to the nontreated control, suggesting that the fluopyram active ingredient was affecting H. glycines directly and was not an inert ingredient in the seed treatment formulation. Hatching, motility, and root penetration experiments also were conducted using only the formulated seed treatments. Exudates collected from ILeVO-treated seed reduced J2 hatching and motility by more than 95% in laboratory assays. Exudates from radicles grown from ILeVO-treated seed reduced hatching in vitro by 48% in one run but had no significant effect in the second run compared with the nontreated control exudates. There also were no consistent effects of radicle exudates, regardless of treatment, on hatching and motility of the J2. ILeVO reduced root penetration of H. glycines J2 at different inoculation densities in a growth chamber experiment. VOTiVO did not affect any of the processes or life stages of the nematode studied. The results of this study indicate that the use of nematode-protectant seed treatments may be useful in controlling H. glycines; however, additional investigations into the precise effects of ILeVO and VOTiVO on H. glycines life processes and in different parts of the soil profile are necessary.

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

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 NH4NO3, KNO3, KCl, MgCl2, and CaCl2. H. glycines was found to be attracted to both NH4NO3 and KNO3, 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.