Assessing Direct and Residual Effects of Cover Crops on the Soybean Cyst Nematode, Heterodera glycines.

Greenhouse experiments were conducted to determine if cover crops directly decrease population densities of the soybean cyst nematode (SCN), Heterodera glycines, and/or have residual effects on reproduction of the nematode on soybean (Glycine max). Population densities of SCN were not significantly decreased by nine cover crop plants or three cover crop mixes compared with a non-planted soil control in a repeated 60-day-long greenhouse experiment. When susceptible soybeans were grown in the soils after cover crop growth, fewer SCN females formed after three annual ryegrass (Lolium multiflorum) cultivars (Bounty, King, and RootMax), the Daikon radish (Raphanus sativus var. longipinnatus) cultivar CCS779, Kodiak mustard (Brassica juncea), and a mix containing cereal rye, crimson clover (Trifolium incarnatum), plus Daikon radish (cultivars not stated) compared with following the non-planted control. In another repeated experiment, cover crops were grown for 56 days in SCN-infested soil in the greenhouse then exposed to Iowa winter conditions for 28 days to simulate winter termination of the plants. One treatment, a cover crop mix containing 'Bounty' annual ryegrass plus 'Enricher' Daikon radish, had a decrease in SCN population density greater than the non-planted control at the end of the experiment. Significantly fewer SCN females formed on soybeans grown after several cover crops, including the three annual ryegrass cultivars that had the suppressive residual effects in the first experiment. In summary, there were no cover crop treatments that consistently decreased SCN population densities across experiments, and only one cover crop treatment in one experiment significantly reduced SCN population densities more than a non-planted soil control. However, there was a somewhat consistent, adverse, residual effect of cover crops on reproduction of SCN on susceptible soybeans after growth of multiple cover crops.

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

Soybeans are an important crop for global food security. Every year, soybean yields are reduced by numerous soybean diseases, particularly the soybean cyst nematode (SCN). It is difficult to visually identify the presence of SCN in the field, let alone its population densities or numbers, as there are no obvious aboveground disease symptoms. The only definitive way to assess SCN population densities is to directly extract the SCN cysts from soil and then extract the eggs from cysts and count them. Extraction is typically conducted in commercial soil analysis laboratories and university plant diagnostic clinics and involves repeated steps of sieving, washing, collecting, grinding, and cleaning. Here we present a robotic instrument to reproduce and automate the functions of the conventional methods to extract nematode cysts from soil and subsequently extract eggs from the recovered nematode cysts. We incorporated mechanisms to actuate the stage system, manipulate positions of individual sieves using the gripper, recover cysts and cyst-sized objects from soil suspended in water, and grind the cysts to release their eggs. All system functions are controlled and operated by a touchscreen interface software. The performance of the robotic instrument is evaluated using soil samples infested with SCN from two farms at different locations and results were comparable to the conventional technique. Our new technology brings the benefits of automation to SCN soil diagnostics, a step towards long-term integrated pest management of this serious soybean pest.

A Mechanistic Approach To Assessing the Potential for Cover Crops To Serve as Trap Crops for the Soybean Cyst Nematode.

The effects of cover crops on the biology of the soybean cyst nematode (SCN; Heterodera glycines) are not well established. It is possible that cover crops may reduce SCN population densities by acting as trap crops. Cover crops with potential to serve as trap crops may stimulate hatching and/or attract hatched SCN juveniles and also may be penetrated by large numbers of nematodes that cannot feed. Experiments were conducted to determine whether root exudates (REs) and soil leachates (SLs) from various cover crop plants affected SCN hatching and chemotaxis and if there were significant differences in SCN juvenile root penetration among different cover crop plant types. In 14-day-long hatching experiments, there was greater SCN hatching in crimson clover (Trifolium incarnatum) REs and SLs than in REs and SLs from all other cover crop treatments in the experiments. No other cover crop REs and SLs significantly affected hatching. In chemotaxis experiments, SCN juveniles were attracted to REs and SLs from annual ryegrass (Lolium multiflorum) and cereal rye (Secale cereale) after 24 h. In greenhouse experiments, significantly more SCN juveniles penetrated the roots of single cultivars of crimson clover, mustard (Brassica juncea), and rapeseed (B. napus) than 11 other cover crop species/cultivars evaluated in the experiment over the course of 20 days. Few SCN juveniles penetrated the roots of annual ryegrass and cereal rye. The results suggest that crimson clover, grown as a cover crop, has the most potential to act as a trap crop for SCN. Cover crop plants may affect SCN biology in ways other than the mechanisms investigated in these experiments.

Effects of ILeVO Seed Treatment on Heterodera glycines Reproduction and Soybean Yield in Small-Plot and Strip-Trial Experiments in Iowa.

ILeVO (fluopyram) is a fungicide seed treatment for soybean sudden death syndrome (SDS) that also has nematicidal activity. ILeVO is sold with a base of insecticide Poncho (clothianidin), nematode-protectant VOTiVO (Bacillus firmus), and Acceleron fungicides (metalaxyl, fluxapyroxad, and pyraclostrobin). Yield and reproduction of the soybean cyst nematode (SCN) (Heterodera glycines) on soybean plants grown from seed treated with ILeVO plus the base were compared with those treated with only the base in 27 small-plot experiments and 12 strip-trial experiments across Iowa from 2015 to 2017. To increase the likelihood that yield results were related to effects on SCN, data were used only from 26 small-plot experiments and 12 strip trials in which symptoms of SDS were low or nonexistent. An SCN reproductive factor (RF) was calculated for each experimental unit by dividing the SCN population density at harvest by the population density at planting. ILeVO significantly reduced SCN RF by 50% in one strip-trial experiment and by 36 to 60% in four small-plot experiments but yields were not increased by ILeVO in any of those five experiments. Soybean yields were 2.8 to 3.7 bushels/acre (bu/ac) (188.3 to 248.8 kg/ha) greater with ILeVO in three small-plot experiments but SCN RF was not reduced in those experiments. Also, yield was 1.9 bu/ac (127.8 kg/ha) greater with ILeVO in one strip-trial experiment in 2016 but SCN samples were not collected at harvest from the study to assess the possible effects of ILeVO on SCN reproduction. When strip-trial data from 2015 and 2016 were combined, there was a small but significant 0.8 bu/ac (52.2 kg/ha) yield increase with ILeVO. Overall, the effects of ILeVO on SCN reproduction and soybean yield were variable in these field studies.

Developing Public-Private Partnerships in Plant Pathology Extension: Case Studies and Opportunities in the United States.

Public-private partnerships (PPPs) can be an effective and advantageous way to accomplish extension and outreach objectives in plant pathology. The greatest opportunities for extension-focused PPPs may be in response to large-scale or emerging disease management concerns or in addressing complex issues that impact agriculture, such as climate change, digital technology, and public perception of science. The most fertile ground for forming PPPs is where the needs and strengths of the public and private sectors are complementary. Developing PPPs depends as much on professional relationships as on technical skills or contracts. Defining and making room for the success of all partners, identifying and addressing barriers to success, and earning and maintaining trust are components that contribute to the effectiveness of PPPs. Case studies in plant pathology demonstrate the positive impact PPPs can have on partners and stakeholders and provide guidance on the formation of PPPs in the future.

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.

QQS orphan gene and its interactor NF-YC4 reduce susceptibility to pathogens and pests.

Enhancing the nutritional quality and disease resistance of crops without sacrificing productivity is a key issue for developing varieties that are valuable to farmers and for simultaneously improving food security and sustainability. Expression of the Arabidopsis thaliana species-specific AtQQS (Qua-Quine Starch) orphan gene or its interactor, NF-YC4 (Nuclear Factor Y, subunit C4), has been shown to increase levels of leaf/seed protein without affecting the growth and yield of agronomic species. Here, we demonstrate that overexpression of AtQQS and NF-YC4 in Arabidopsis and soybean enhances resistance/reduces susceptibility to viruses, bacteria, fungi, aphids and soybean cyst nematodes. A series of Arabidopsis mutants in starch metabolism were used to explore the relationships between QQS expression, carbon and nitrogen partitioning, and defense. The enhanced basal defenses mediated by QQS were independent of changes in protein/carbohydrate composition of the plants. We demonstrate that either AtQQS or NF-YC4 overexpression in Arabidopsis and in soybean reduces susceptibility of these plants to pathogens/pests. Transgenic soybean lines overexpressing NF-YC4 produce seeds with increased protein while maintaining healthy growth. Pull-down studies reveal that QQS interacts with human NF-YC, as well as with Arabidopsis NF-YC4, and indicate two QQS binding sites near the NF-YC-histone-binding domain. A new model for QQS interaction with NF-YC is speculated. Our findings illustrate the potential of QQS and NF-YC4 to increase protein and improve defensive traits in crops, overcoming the normal growth-defense trade-offs.

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

Nematicidal seed treatments are a relatively new strategy for managing plant-parasitic nematodes in row crops. Two such seed treatments, Avicta (abamectin) and Clariva (Pasteuria nishizawae), are marketed by Syngenta for use against Heterodera glycines in soybean production in the upper Midwest. The specific effects of these seed treatments on the biology of the nematode have not been previously reported. The effects of Avicta and Clariva on H. glycines hatching, movement, attraction, penetration, development, and reproduction were determined in controlled-environment experiments. Avicta inhibited juvenile movement and penetration at the seed depth and 3 cm below the seed. Clariva inhibited juvenile movement and penetration 3 and 5 cm below the seed and nematode development within the roots of young plants. Both seed treatments affected nematodes in 10- and 20-day-old plants, but effects were not detected on nematodes developing in older plants (30 and 60 days) with larger root systems. These results provide details of the specific mechanisms of early-season protection provided by Avicta and Clariva seed treatments.

A deep learning framework to discern and count microscopic nematode eggs.

In order to identify and control the menace of destructive pests via microscopic image-based identification state-of-the art deep learning architecture is demonstrated on the parasitic worm, the soybean cyst nematode (SCN), Heterodera glycines. Soybean yield loss is negatively correlated with the density of SCN eggs that are present in the soil. While there has been progress in automating extraction of egg-filled cysts and eggs from soil samples counting SCN eggs obtained from soil samples using computer vision techniques has proven to be an extremely difficult challenge. Here we show that a deep learning architecture developed for rare object identification in clutter-filled images can identify and count the SCN eggs. The architecture is trained with expert-labeled data to effectively build a machine learning model for quantifying SCN eggs via microscopic image analysis. We show dramatic improvements in the quantification time of eggs while maintaining human-level accuracy and avoiding inter-rater and intra-rater variabilities. The nematode eggs are correctly identified even in complex, debris-filled images that are often difficult for experts to identify quickly. Our results illustrate the remarkable promise of applying deep learning approaches to phenotyping for pest assessment and management.

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.

Interactions of effects of host plant resistance and seed treatments on soybean aphid (Aphis glycines Matsumura) and soybean cyst nematode (Heterodera glycines Ichinohe).

BACKGROUND: The soybean cyst nematode, Heterodera glycines, and soybean aphid, Aphis glycines, are invasive, widespread and economically important pests of soybean, Glycine max, in North America. Management of these pests relies primarily on use of pesticides and soybean germplasm with genetic resistance. A 3-year field study and complementary greenhouse experiment were conducted to determine the benefits of host plant resistance (HPR) and pesticidal seed treatments for managing pest populations and preserving soybean yield. RESULTS: Host plant resistance significantly decreased the abundance of A. glycines and, in most study sites, suppressed H. glycines. Neonicotinoid seed treatment reduced A. glycines abundance on the cultivar that was susceptible to both aphids and nematodes, but abamectin nematicide seed treatment had no effect on H. glycines populations in the field or greenhouse. CONCLUSION: These results suggest that the seed treatments included in our experiments may suppress pests, but not consistently for all soybean cultivars or study sites. Ultimately, HPR more consistently reduced pest numbers compared with the use of pesticidal seed treatments. The planting of HPR cultivars should be a primary tool for integrated pest management of both soybean pests. © 2017 Society of Chemical Industry.

Validation of a hairy roots system to study soybean-soybean aphid interactions.

The soybean aphid (Aphis glycines) is one of the main insect pests of soybean (Glycine max) worldwide. Genomics approaches have provided important data on transcriptome changes, both in the insect and in the plant, in response to the plant-aphid interaction. However, the difficulties to transform soybean and to rear soybean aphid on artificial media have hindered our ability to systematically test the function of genes identified by those analyses as mediators of plant resistance to the insect. An efficient approach to produce transgenic soybean material is the production of transformed hairy roots using Agrobacterium rhizogenes; however, soybean aphids colonize leaves or stems and thus this approach has not been utilized. Here, we developed a hairy root system that allowed effective aphid feeding. We show that this system supports aphid performance similar to that observed in leaves. The use of hairy roots to study plant resistance is validated by experiments showing that roots generated from cotyledons of resistant lines carrying the Rag1 or Rag2 resistance genes are also resistant to aphid feeding, while related susceptible lines are not. Our results demonstrate that hairy roots are a good system to study soybean aphid-soybean interactions, providing a quick and effective method that could be used for functional analysis of the resistance response to this insect.

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.

Soybean Aphid Infestation Induces Changes in Fatty Acid Metabolism in Soybean.

The soybean aphid (Aphis glycines Matsumura) is one of the most important insect pests of soybeans in the North-central region of the US. It has been hypothesized that aphids avoid effective defenses by inhibition of jasmonate-regulated plant responses. Given the role fatty acids play in jasmonate-induced plant defenses, we analyzed the fatty acid profile of soybean leaves and seeds from aphid-infested plants. Aphid infestation reduced levels of polyunsaturated fatty acids in leaves with a concomitant increase in palmitic acid. In seeds, a reduction in polyunsaturated fatty acids was associated with an increase in stearic acid and oleic acid. Soybean plants challenged with the brown stem rot fungus or with soybean cyst nematodes did not present changes in fatty acid levels in leaves or seeds, indicating that the changes induced by aphids are not a general response to pests. One of the polyunsaturated fatty acids, linolenic acid, is the precursor of jasmonate; thus, these changes in fatty acid metabolism may be examples of "metabolic hijacking" by the aphid to avoid the induction of effective defenses. Based on the changes in fatty acid levels observed in seeds and leaves, we hypothesize that aphids potentially induce interference in the fatty acid desaturation pathway, likely reducing FAD2 and FAD6 activity that leads to a reduction in polyunsaturated fatty acids. Our data support the idea that aphids block jasmonate-dependent defenses by reduction of the hormone precursor.

Aboveground feeding by soybean aphid, Aphis glycines, affects soybean cyst nematode, Heterodera glycines, reproduction belowground.

Heterodera glycines is a cyst nematode that causes significant lost soybean yield in the U.S. Recent studies observed the aphid Aphis glycines and H. glycines interacting via their shared host, soybean, Glycine max. A greenhouse experiment was conducted to discern the effect of A. glycines feeding on H. glycines reproduction. An H. glycines-susceptible cultivar, Kenwood 94, and a resistant cultivar, Dekalb 27-52, were grown in H. glycines-infested soil for 30 and 60 d. Ten days after planting, plants were infested with either zero, five, or ten aphids. At 30 and 60 d, the number of H. glycines females and cysts (dead females) and the number of eggs within were counted. In general, H. glycines were less abundant on the resistant than the susceptible cultivar, and H. glycines abundance increased from 30 to 60 d. At 30 d, 33% more H. glycines females and eggs were produced on the resistant cultivar in the ten-aphid treatment compared to the zero-aphid treatment. However, at 30 d the susceptible cultivar had 50% fewer H. glycines females and eggs when infested with ten aphids. At 60 d, numbers of H. glycines females and cysts and numbers of eggs on the resistant cultivar were unaffected by A. glycines feeding, while numbers of both were decreased by A. glycines on the susceptible cultivar. These results indicate that A. glycines feeding improves the quality of soybean as a host for H. glycines, but at higher herbivore population densities, this effect is offset by a decrease in resource quantity.