Investigating the Interactions Between Cover Crops and the Soybean Cyst Nematode Through Lab, Greenhouse, and Field Studies

Iowa State University Capstones, Theses and Graduate Theses and Dissertations Dissertations

2019

Investigating the interactions between cover crops and the soybean cyst nematode through lab, greenhouse, and field studies

Chelsea Jean Harbach Iowa State University

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Recommended Citation Harbach, Chelsea Jean, "Investigating the interactions between cover crops and the soybean cyst nematode through lab, greenhouse, and field studies" (2019). Graduate Theses and Dissertations. 17459. https://lib.dr.iastate.edu/etd/17459

This Dissertation is brought to you for free and open access by the Iowa State University Capstones, Theses and Dissertations at Iowa State University Digital Repository. It has been accepted for inclusion in Graduate Theses and Dissertations by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. Investigating the interactions between cover crops and the soybean cyst nematode through lab, greenhouse, and field studies

Chelsea Jean Harbach

A dissertation submitted to the graduate faculty

in partial fulfillment of the requirements for the degree of

DOCTOR OF PHILOSOPHY

Major: Plant Pathology

Program of Study Committee: Gregory L. Tylka, Major Professor Leonor F. Leandro Daren S. Mueller Thomas C. Kaspar Matthew J. Helmers

The student author, whose presentation of the scholarship herein was approved by the program of study committee is solely responsible for the content of this dissertation. The Graduate College will ensure this dissertation is globally accessible and will not permit alterations after a degree is conferred.

Iowa State University

Ames, Iowa

2019

DEDICATION

Dedicated in loving memory of Willard (Bill) Harbach April 3, 1943 – May 10, 2019.

I dedicate this work to my Grandpa, Bill Harbach. Grandpa, you may no longer with us on this earth, but your legacy lives on. You started your farm from nothing and nurtured it to grow into what it is today. Your passion helped inspire my dad to follow in your footsteps. It was your love for me that helped push me back to agriculture during my undergraduate studies.

Without that push, I would never have found my professional passion in life, plant pathology.

Thank you for setting such a strong example for me, Grandpa. I love and miss you every second of every day.

I would also like to dedicate this to my parents, Heath and Barb, and my Meema, for your unyielding love and support throughout all of my schooling. To Melissa Goodwin, my partner, thank you for your patience and love throughout my studies. And last but not least, to my three- legged corgi boy Charles Avocado Van Gogh. It has been a rough ride for us, little buddy, but thank you for being so resilient and hanging around to be a part of my support system.

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TABLE OF CONTENTS

Page

ACKNOLEDGEMENTS...... v

ABSTRACT...... vi

CHAPTER 1. GENERAL INTRODUCTION AND LITERATURE REVIEW...... 1 Dissertation Organization...... 1 Introduction and Literature Review...... 1 Soybean Cyst Nematode...... 1 H. glycines Biology...... 2 SCN Management in Conventional Corn-Soybean Systems...... 4 Host Resistance...... 4 Seed Treatments...... 5 Rotation with Nonhost Crops...... 5 History and Resurgence of Cover Crops in the United States...... 6 Alternative Cropping System Effects on Other Plant-Parasitic Nematodes...... 7 Potential for Cover Crops Impacting SCN...... 9 Overview and Objectives...... 13 Literature Cited...... 14

CHAPTER 2. DETERMINING THE IMPACTS OF COVER CROPS ON SOYBEAN CYST NEMATODE HATCHING, CHEMOTAXIS, AND ROOT PENETRATION...... 20 Abstract...... 20 Introduction...... 21 Materials and Methods...... 23 Collection of Root Exudates and Soil Leachates...... 23 Hatching Experiments...... 24 Chemotaxis Experiments...... 26 Root Penetration Experiments...... 27 Data Analyses...... 28 Results...... 30 Hatching Experiments...... 30 Chemotaxis Experiments...... 30 Root Penetration Experiments...... 31 Discussion...... 31 Literature Cited...... 34

CHAPTER 3. ASSESSING THE DIRECT AND RESIDUAL EFFECTS OF COVER CROPS ON SOYBEAN CYST NEMATODE POPULATION DENSITIES...... 42 Abstract...... 42 Introduction...... 43 Materials and Methods...... 45 Experiments to Assess Direct Effects of Cover Crops on SCN Population Densities...... 45 Experiments to Assess the Residual Effects of Cover Crops on SCN Reproduction...... 47 Experiments to Assess the Effect of Winterkilled Cover Crops on SCN Population Densities...... 48 Data Analyses...... 49 Results...... 50 Direct Effects of Cover Crops on SCN Population Densities in Greenhouse Conditions...... 50 Residual Effects of Cover Crops on SCN Reproduction in Greenhouse Conditions...... 50 Direct and Residual Effects of Winterkilled on Cover Crops on SCN Population Densities...... 51 Discussion...... 51 Literature Cited...... 54

CHATPER 4. THE EFFECTS OF COVER CROPS ON SOYBEAN CYST NEMATODE POPULATION DENSITIES IN MULTI-YEAR, SMALL-PLOT FIELD EXPERIMENTS IN IOWA...... 61 Abstract...... 61 Introduction...... 62 Materials and Methods...... 63 Soil Processing...... 65 Data Analyses...... 66 Results...... 66 Discussion...... 67 Literature Cited...... 71

CHAPTER 5. GENERAL CONCLUSION...... 78

APPENDIX: DETERMINING HIGH OR LOW SOYBEAN CYST NEMATODE POPULATION DENSITY LEVELS IMPACT ABILITY TO OBSERVE DIFFERENCES IN POPULATION CHANGE FACTOR DUE TO COVER CROPS...... 80 Introduction...... 80 Materials and Methods...... 80 Results...... 82 Discussion...... 82 Literature Cited...... 82

ACKNOWLEDGEMENTS

First and foremost, a most sincere note of gratitude to my adviser, Gregory Tylka, for the offer of a lifetime to work and grow creatively as a scientist in his laboratory, providing his mentorship, sharing his expertise, and providing me countless opportunities to continue to flourish as a plant pathologist. Thank you to the faculty on my committee for your time and guidance: Leonor Leandro, Daren Mueller, Matthew Helmers, and Thomas Kaspar. Much of my field work was done with help from the Tylka lab staff, Chris Marett, Mark Mullaney, Greg

Gebhart, David Soh, and numerous undergraduate students that assisted me throughout, especially Corey Tjaden. I am so grateful for the support of my lab-mates, both past and present, including Augustine Beeman, Jared Jensen, EB Wlezien, Jefferson Barizon, and Monica

Pennewitt. And an immense thank you to my friend and mentor, Kaitlyn Bissonnette.

Thank you to those that collaborated with me professionally including the Iowa Soybean

Association and those that donated seed for my research including Kim Davidson (Davidson

Commodities, Mighty Mustard), Tim Gioffredi (LaCrosse Seed), Brian Weiland (Saddle Butte

Ag. Inc.), Karl Dallfeld (Prairie Creek Seed), and Jonathan Rupert (Smith Seed Services). Thank you to the USDA North Central SARE graduate student grant program for help in funding my research. And lastly, thank you to the late Mike Plumer, whose data helped inspire my research.

ABSTRACT

Throughout the past decade, the acres on which cover crops have been planted in agricultural systems in the United States has increased. In traditional row cropping, cover crops are sown shortly before or after the harvest of corn or soybean and grown through the fall to provide ground cover for approximately one third of the year during which the soil would otherwise be bare. Cover crops have many known environmental benefits, but the effects they may have on soybean cyst nematode (SCN), Heterodera glycines, are not well established. This dissertation describes experiments conducted i) in the laboratory and greenhouse to determine if cover crops have potential to serve as trap crops, ii) in the greenhouse to determine whether cover crops affect SCN population densities under controlled conditions and iii) in the field to assess how cover crops affect SCN population densities in multi-year, multi-location and rotation situations.

The first set of experiments assessed the possibility that cover crop species may act as trap crops for SCN. A good candidate for an SCN trap crop might stimulate SCN hatching, attract hatched SCN juveniles, and/or would be infected by many nematodes. Root exudates and soil leachates (RE/SL) were collected from cover crop plants and used to test their effects on

SCN hatching and chemotaxis. The hatching of SCN in crimson clover RE/SL was greater than in all other cover crop treatments as well as in the unplanted control. There were no cover crop

RE/SL that had notable effects on SCN chemotaxis. In greenhouse experiments where root penetration by SCN was assessed, more SCN juveniles were recovered from roots of crimson clover than most other cover crop treatments. In total, these results suggest crimson clover has the most potential to act as a trap crop for SCN. vii

A set of greenhouse experiments were conducted to determine if cover crops affect SCN population densities and subsequent SCN reproduction. After sixty days of growth by different cover crops, the SCN population density decreased numerically in all treatments. There were no differences in the amount of SCN population density decrease over the duration of the experiment for cover crop treatments compared to the non-planted soil control. When susceptible soybean plants were grown in leftover soil from this experiment, there were significantly fewer

SCN females that developed on soybean roots following annual ryegrass 1, annual ryegrass 2, annual ryegrass 3, daikon radish, mustard 1, mix 1, and the tomato control compared to following the non-planted control. And in an experiment where cover crops were grown for 56 days followed by a 28-day exposure to Iowa winter conditions, there were no differences in SCN population density decreases for all cover crop treatments compared to the non-planted soil control. On soybeans grown in soil following cover crop growth, there were fewer SCN females that formed on soybean roots following seven of the treatments included all three annual ryegrass treatments, compared to the following the non-planted soil control. Overall, these results indicate that there may be an adverse residual effect of at least some cover crops on SCN reproduction.

To expand the research to real-world field environments, experiments were conducted in

2016-2017 and 2017-2018 at the Iowa State University Muscatine Island Research Farm in

Fruitland, Iowa, and at the Northern Research Farm in Kanawha, Iowa, with two experiments at each location designated by the annual crop rotation: corn-soybean and soybean-corn. Cover crop seeds were broadcasted into standing corn and soybean in late summer. Soil samples were collected at the time of cover crop seeding, again in late fall prior to a hard freeze, and once more in the spring prior to planting of corn or soybean to determine changes in SCN population densities each year. After two complete years of these experiments, there were no significant viii differences in the SCN population density changes observed among all treatments, sampling intervals, fields, locations, or years. Furthermore, there were no significant differences in the

PCF means for any cover crop treatment compared to the non-planted control. None of the cover crops decreased SCN population densities under the conditions of these field experiments.

The high spatial variability of SCN both within and among small plots may explain why the effects of cover crops detected in greenhouse and laboratory experiments were not apparent in the field. More work on cover crops, especially those with promise as trap crops, is warranted under highly-controlled conditions.

CHAPTER 1. INTRODUCTION AND LITERATURE REVIEW

Dissertation Organization

This dissertation will be organized into five chapters. The first chapter includes an introduction and literature review for the soybean cyst nematode, conventional management, and what we know about the cover crop interactions with soybean cyst nematodes thus far. The second, third, and fourth chapters outline the methods and results for different types of experiments examining the research question. The fifth chapter is an overall summary of the findings from the research.

Introduction and Literature Review

Cover crops have been extensively studied from an agronomic standpoint and are known to prevent soil erosion and mitigate nutrient leaching (Clark 2007). As a result of these benefits, the use of cover crops has increased throughout the United States over the past five years (CTIC

2017). While much is known about the conservation benefits of growing cover crops, research on the interactions of cover crops with other aspects of the complex crop production systems, such as plant pathogens, is still fairly new.

A pathogen of particular interest in soybean production systems is Heterodera glycines

(Ichinohe), the soybean cyst nematode (SCN). This chapter will review the role of SCN on soybean production in the United States, the history of cover crops and their effects on other plant-parasitic nematodes, and the potential for cover crops to affect SCN population densities.

Soybean Cyst Nematode

While it is suspected that the SCN was known to exist in third-century China, it was not scientifically identified until 1915 in Japan and was later formally characterized in 1952 2

(Ichinohe 1952). The soybean pest was first identified in the United States in 1954 (Winstead et al. 1955). Since its initial discovery in the United States, SCN has steadily spread throughout most of the soybean-producing areas of the country. As of 2017, SCN has been found in every county in Illinois and Iowa, the two top soybean-producing states in the United States (Tylka and

Marett 2017). The SCN has been consistently estimated to be the top yield-suppressing pathogen in soybean production in the United States for the past 20 years (Koenning and Wrather, 2009;

Wrather and Koenning, 2010; Allen et al. 2017). The most effective method of control for SCN is prevention, i.e. avoid the introduction of the nematodes into a non-infested field Once SCN is present in a field, it is nearly impossible to eradicate from the soil, and management should be focused on suppressing population density growth. The biology of H. glycines can make it particularly difficult to manage.

H. glycines Biology

The “cyst” in “soybean cyst nematode” refers to the hardened cuticle of what was the female body, and it can contain hundreds of eggs (Davis and Tylka 2005). The cysts can exist in the soil, serving as a protective barrier for the eggs, for many years. The first molt for SCN juveniles occurs within the egg, resulting in a second-stage juvenile (J2) hatching from the egg.

The J2s will hatch from their eggs in favorable conditions, most importantly soil temperature, presence of roots, and time (Hill and Schmitt 1989; Masler and Rogers 2011;

Niblack 2005; Turner and Subbotin 2013). However, some J2s, mostly from eggs in the gelatinous matrix, hatch constitutively (Thompson and Tylka 1997). After hatching, J2s use chemotaxis to move towards roots following a gradient of root exudates (Papademetriou and

Bone 1983). Once arrived at a root, the SCN juvenile uses cell-wall-degrading enzymes excreted through the stylet to break down plant tissue and migrate through the cortex to the root 3 vasculature (Smant et al. 1998). Upon arrival at the vascular stele, the nematode pushes its stylet through the cell wall and begins secreting effectors that manipulate the plant and break down the plant the cell walls of neighboring cells,, resulting in one, large, multi-nucleate, photosynthetic sink cell functioning as a feeding site for the juvenile, called a syncytium (Bohlmann and

Sobczak 2014). The juvenile forms a feeding tube in the cytoplasm of the cell to absorb nutrients from the syncytium (Wyss and Grundler 1992). Here, the nematode remains for the rest of its life continuing through a second and third molt while sedentary and advancing to a fourth-stage juvenile.

The final molt from fourth-stage juvenile (J4) to mature adult reveals the sexual dimorphism of adult SCN (Davis and Tylka, 2000) as the female begins to swell as ovaries develop and the male reverts to a vermiform shape that is three times the length of the J2, folded in the J4 cuticle. The adult male has a weak stylet and does not feed. Instead, the male exits the root and uses chemotaxis (Jaffe et al. 1989) to migrate towards SCN females that have grown so large that they are accessible on the outside surface of the root (Davis and Tylka 2000). The SCN females can be mated by multiple males, resulting in an offspring with greater genetic diversity than if there was only one mating event (Triantaphyllou and Esbenshade 1990). However, most matings are likely between siblings or half-siblings, which does not appear to hinder the reproductive capacity of H. glycines populations (Luedders 1985). As the female continues to produce more eggs, eventually the ovaries and fertilized eggs crush the nematode’s organs resulting in death. The female will produce hundreds of eggs, some in a gelatinous matrix outside of her body, but most are protected by the cuticle of her body (Davis and Tylka 2000).

As the cuticle ages, it hardens and turns brown, and the cyst dislodges from the soybean root and will exist in the soil for many years. 4

This entire life cycle can be completed in less 21 days under gnotobiotic conditions

(Lauritis et al. 1983), which could allow for multiple generations of SCN to occur in a single growing season. In Iowa, soybean seeds are usually planted in May and harvested in late

September or October resulting in a growing season of 140 days or more. There could be 3 to 4 generations per growing season and even more in the southern United States. If this soybean pest goes unnoticed or unchecked, the population density could increase dramatically in a single growing season.

SCN Management in Conventional Corn-Soybean Systems

Host Resistance

Shortly after identifying SCN in the United States, researchers screened soybean germplasm to determine if there were any plant introductions (PI) that already exhibited resistance to SCN. Ross and Brim (1957) evaluated more than 2,800 PIs and identified four entries on which few (less than ten) SCN females developed, including Peking.

Currently there is little diversity in sources of resistance that exist in commercially developed soybean genotypes. For example, approximately 95% of SCN-resistant cultivars in

Iowa contain resistance genes from PI88788 (Tylka and Mullaney 2018). As a result of prolonged use of a single source of SCN resistance in commercial soybean cultivars, an increase in SCN reproduction has been seen on cultivars with resistance derived from PI 88788

(McCarville et al. 2017; Howland et al. 2018). To make issues more complicated, researchers discovered that the efficacy SCN resistance derived from PI 88788 is mediated by a quantitative trait locus, particularly the number of copies of Rhg1 within the locus (Cook et al. 2012).

Soybean genotypes with greater SCN resistance have greater copy number variation, and yet there is no reporting on the number of copies of Rhg1 for commercially available SCN-resistant 5 cultivars derived from PI 88788. In reality, the PI 88788 resistance is actually conferred on an unknown gradient.

Seed Treatments

Though nematicidal fumigants are known to decrease the SCN population densities in the soil (Sasser and Uzzell, Jr. 1991), it is economically and ecologically irresponsible to resort to such methods in soybean production fields that span millions of acres throughout the United

States (Davis and Tylka 2000). Over the past decade, researchers have identified new chemicals and biological control methods that can be applied to soybean seed as seed treatments in attempt to mitigate population density growth of SCN. Applying chemicals or biocontrol agents to seed helps diminish the risk of affecting non-target microorganisms, limits the volume of compounds needed, and decreases the exposure to potentially toxic material for farmers (Munkvold et al.

2014). The seed treatments that are currently available to farmers for SCN management have a wide variety of known and unknown activity against the pest (Bissonnette and Tylka 2017).

However, the overall efficacy of seed treatments is inconsistent when studied in the field

(Bissonnette et al. 2018) and likely depends on the environment during the growing season, the

SCN pressure, and the soybean cultivar planted. The number and diversity of seed treatments for

SCN management continues to increase and need evaluation to determine their efficacy.

Rotation with Nonhost Crops

There has been a renewed effort to incorporate an additional source of SCN-resistance into commercial soybean cultivars recently. Meanwhile, rotation with non-host crops remains another crucial method in which to mitigate the population density growth of SCN. Due to the apparent coevolution of Heterodera glycines and Glycine max, SCN has a relatively limited host range including other leguminous crops and some weed species (Poromarto et al. 2015; Creech 6 et al. 2007; Venkatesh et al. 2000). However, SCN juveniles can enter the roots of any plant regardless of host suitability and will enter corn roots and subsequently starve (Warnke et al.

2008) leading to a decrease in population density in years that fields are rotated to a non-host crop. There is interest in the viability of using non-host cover crops to help manage SCN population densities as well.

History and Resurgence of Cover Crops in the United States

The implementation of cover crops into cropping systems throughout the United States has been steadily increasing over the past half-decade (CTIC 2017). There are multiple reasons that farmers are adopting this practice at an increasing rate.

Cover cropping is a historical practice that can be traced back to the first president of the

United States, George Washington. President Washington knew the value of “growing crops to eat and sell” and “crops grown to replenish the soil” (Groff 2015). For centuries, farmers have known about the benefits of cover cropping in order to provide some nutrient return for the soil.

However, following World War II, the technology to synthesize nitrogen fertilizers led to an overall decrease in the use of cover crops in the following years (Groff 2015). The rate at which nitrogen fertilizers were used increased tremendously, especially in the corn belt from the 1940s to the 2010s (Cao et al. 2018). Researchers have observed the resulting impacts of nutrient leaching into ground water and watersheds (Billen et al. 2013), which can subsequently have detrimental impacts on water ecosystems and human health. For example, the hypoxic zone in the Gulf of Mexico waxes and wanes as a result of leached agricultural nutrients (CENR, 2000) and contaminating municipal drinking water systems (Nolan et al. 1998). In some cases, such as the Des Moines Water Works, there have been lawsuits filed against farmers for mismanagement of fertilizers resulting in contaminated drinking water (Essman 2017). 7

Farmers, in need of a management practice that can help them mitigate soil leaching of nutrients, have increasingly started to implement cover crops. Cover crops are known for their ability to capture nutrients and mitigate leaching, prevent soil erosion, and increase the soil organic carbon available to the plant (Clark, 2007). While the agronomic benefits of cover crops are well established, what has not been examined in-depth is the interaction that cover crops have with plant pathogens.

Alternative Cropping System Effects on Other Plant-Parasitic Nematodes

Generally, the efficacy of cover crops in mitigating plant-parasitic nematode population densities can be determined by evaluating one of several potential mechanisms through which the plant can decrease populations (Niblack and Chen 2004). First is the idea of a non-host plant also serving as a trap crop in which the nematode enters the roots and dies. Second, cover crops could produce toxic allelochemicals that directly kill nematodes or eggs in the soil. Third, cover crops could release inhibitory allelochemicals that indirectly affect nematode or egg populations in the soil by decreasing the overall health or mobility of the nematode. And lastly, cover crops may produce root exudates that differentially induce nematode egg hatch during a period in which there are no suitable host roots in the soil, subsequently starving the nematode larvae.

There are some cases in which cover crops have reduced the population densities of plant-parasitic nematodes. In 1938, researchers reported that certain cultivars of marigold

(Tagetes erecta), called “Mexican marigold”, showed high resistance to the root-knot nematode,

RKN (Meloidogyne spp.) (Tyler 1938). Three years later, although not the primary purpose of the study, Steiner (1941) found that a large number of RKN that entered the roots of various

Tagetes spp. never developed to maturity within the roots, perhaps the first report of a trap crop for plant-parasitic nematodes. 8

Since early first reports of the adverse effects on RKN by marigold species, much research has been done with marigolds to observe the impacts on other plant-parasitic nematodes as well as specificity of effects among species of both Tagetes spp. and Meloidogyne spp. The

French marigold (T. patula) was shown to suppress lesion nematode species Pratylenchus penetrans and P. pratensis (Oostenbrink 1960) as well as four RKN species, M. arenaria, M. incognita, M. javanica, and M. hapla (Suatmadji, 1969). Further research by Suatmadji (1969) revealed that Mexican marigold cultivars suppressed the previously mentioned Meloidogyne species except for M. hapla. Suatmadji microscopically observed the roots of the marigold species to assess the development of RKN and found little or no development of the juveniles past the second stage. French marigold was also a non-host for and limited the development of reniform nematode (Rotylenchulus reniformis) in Hawaii (Caswell et al. 1991). A comprehensive review on marigold effects on plant-parasitic nematode species was published by Hooks et al.

(2010).

Some brassica species may serve as trap crops, as well. Research by Hafez and

Sundararaj (2009) found that oilseed radish cultivars Colonel and Ramses significantly reduce sugar beet cyst nematode (Heterodera schacthii) population densities compared to mustard, by serving as a trap crop. However, Bates et al. (2005) suspect that these oilseed radish cover crops could serve as an alternative host for RKN and lesion nematodes.

While marigold species have a well-established history acting as a trap crop for endoparasitic nematodes, they are largely integrated into cropping systems by inter-cropping, double cropping, or as a rotation crop (Hooks et al. 2010). Crotalaria spp. (rattlepod) is a genus of flower plants in the Fabaceae family that has a potential for implementation as a cover crop to mitigate problems caused by various plant-parasitic nematodes including sedentary endoparasitic 9

RKN species and reniform nematode, and some migratory ectoparasitic species from the genera

Belonolaimus (sting nematode), Paratrichodorus (stubby-root nematode), Xiphinema (dagger nematode), and Radopholus (burrowing nematode) (Wang et al. 2002). One of the most widely available species of Crotalaria is C. juncea, or Sunn Hemp. This species is primarily grown in tropical to sub-tropical environments, thus any potential for detrimental effects on plant-parasitic nematodes would be limited to the suitable hardiness zone. There is only one species of

Crotalaria that is native to Iowa, C. sagittalis (plants.usda.gov), and to date there is no report of effects of this plant on plant-parasitic nematodes.

In general, there is a lot of interest in the potential effects of Cruciferous cover crops on plant-parasitic nematodes for the fact that brassica species contain various glucosinolate compounds (species dependent) that release isothiocyanates upon hydrolysis by the myrosinase molecules compartmentalized in the plant vacuole during decomposition of plant cells

(Antonious et al. 2009). The volatile isothiocyanate compounds have nematicidal properties first confirmed on potato cyst nematode (Globodera spp.) by Smedley (1939).

Historically, the above-described mechanisms of population density reduction by cover crops have been easiest to assess in populations of nematodes from species that act as a sedentary-endoparasite, such as SCN. However, there is little to no conclusive data regarding the interaction between cover crops and SCN populations.

Potential for Cover Crops Impacting SCN

With the increased implementation of cover crops and a SCN problem that is increasingly difficult to manage, there is a renewed interest in the notion that cover crops could be a part of the approach to managing SCN population densities. Previously published data on this topic are inconclusive, inconsistent, or difficult to apply to field conditions. Perhaps the most circulated 10 and cited data come from an un-published Extension presentation on experiments conducted in

Illinois in the early 2000s. M. Plumer from University of Illinois presented data showing the use of cereal rye, Secale cereale (cv. Aroostook), and annual ryegrass, Lolium multiflorum (cv.

Bounty), as a cover crop significantly reduced SCN population densities and in one instance completely eradicated the pest from a soybean field (M. Plumer, personal communication).

These data were never published in a peer-reviewed scientific journal. Furthermore, the experiments were conducted in fields with relatively low initial SCN population densities and there was no accounting for a typical corn/soybean rotation (personal communication).

Other published studies examined the interaction between cover crops and SCN.

Kobayashi-Leonel et al. (2017) determined that non-leguminous cover crop species, including cruciferous and grass species, do not support any SCN reproduction but that some leguminous species support a low amount of reproduction (less than five females per plant root). The results of these experiments eliminate any serious concern that cover crops could dramatically increase

SCN population densities by acting as inadvertent hosts.

A field study conducted in Minnesota in which cover crop species were inter-seeded within a standing soybean crop found a decreased SCN population density in the alfalfa and red clover treatments. However, this trend was not consistent for all years and location (Chen et al.

2006). In another study in Minnesota, Miller et al. (2006) observed no significant SCN population density reduction by using “rotation crops” compared to the non-cover crop, non-host control corn (Zea mays) or even a corn with a cereal rye cover crop treatment. Rotation crops in this study included leguminous cover crops alfalfa and red clover. In a field studies from Illinois studying the effects of various cover crop treatments on soybean pathogens, researchers found that rapeseed plots most consistently had lower SCN population densities compared to other 11 treatments in four out of twelve site-years (Wen et al. 2017). However, these field experiments were not designed for monitoring SCN population densities over time and the response variable that was used for comparison was one soil sample collected from each plot in the spring of each year in a random zig-zag pattern, meaning that there was no soil sample to compare the initial population density to prior to application of the cover crop treatment. Furthermore, the population densities in most of these plots that were used in these experiments were very low

(range: 0-2,640, median: 125, mean: 351 eggs/100 cm3). There studies published do not provide adequate data to draw reliable conclusions on the viability of cover crops to help mitigate the

SCN problem in soybean fields in the Midwest.

There are multiple published greenhouse studies that assessed the effect of cover crop species on SCN population densities. In Warnke et al. (2006), studies were conducted with 46 different crop treatments, including corn and BT (Bacillus thurengiensis-transformed) corn in order to observe effects on the percent change factor, calculated as (final population density):(initial population density), to detect changes in population density over time.

Researchers observed the percent change factor (PCF) was not significantly lower for any cover crop treatment compared to the non-cover crop control, corn. Riga et al. (2001) grew cover crop treatments for two months and harvested whole plant masses, chopped them up, and incorporated them into an artificial growth medium, followed by infestation with 300 SCN juveniles. Five days after infestation, susceptible soybeans were transplanted into the pots and grown for 56 days after which the nematodes were extracted from soybean roots as well as the soil surrounding the soybean roots. They found that there were significantly fewer SCN in soil surrounding the roots grown in soil incorporated with several leguminous species, annual ryegrass, and perennial ryegrass compared to the soybean and corn controls (Riga et al. 2001). 12

However, the results of these experiments may not apply to effects in soybean fields because the experiments used freshly hatch J2s and an artificial growth medium.

Limited research has been conducted to assess the effects of various cover crops to serve as trap crops for SCN. Warnke et al. (2008) conducted an experiment where they infested experimental units containing five cover crop species, corn, or soybean with 2,000 freshly hatched second-stage juveniles and later observed the number of nematodes that entered the plant roots using a root-staining methodology from Byrd et al. (1983). Results from this study did not detect any significant differences among plant species (Warnke et al. 2008). To my knowledge, this is the only published study that assessed if cover crop species serve as a trap crop for SCN. However, this experiment used freshly hatched juveniles, which is not entirely relatable to field conditions.

There have been some laboratory studies that investigated whether certain cover crops positively stimulate the hatch of SCN eggs. Warnke et al. (2008) conducted experiments with root exudates and soil leachates. There were some significant differences among treatments after fourteen days of exposure to treatments, but the overall cumulative percent hatch for root exudate treatments was very low (<8.2%). In general, the results from these hatch experiments were inconsistent and difficult to draw conclusions. Another study assessed SCN hatching as affected by cover crop root exudates collected from plants grown in pearlite (Riga et al. 2001). In these experiments, significantly more SCN eggs hatched in root exudates from white clover and annual ryegrass than in the soybean root exudate control. However, this study had very few SCN eggs per experimental unit (n = 20), and there was no non-cover crop, non-host control nor positive hatch stimulating control included in these experiments. 13

Some researchers have conducted experiments to assess the effects of cover crops or their by-products on the biology and behavior of SCN. When brassica species decompose and the glucosinolates are hydrolyzed by myrosinase, the resulting isothiocyanate molecule will vary from species to species (Oregon State University). The most commonly used brassica cover crops, radish and mustard, result in allyl-isothiocyanate (Oregon State University). Schroeder and MacGuidwin (2010) found that there are differential impacts on SCN mortality and movement depending on the type of isothiocyanate molecule used as a treatment. The allyl- isothiocyanate treatment was least effective at killing or inhibiting spontaneous movement of

SCN compared to benzyl- and phenyl- isothiocyanates (2010). In an experiment to observe how fresh plant extracts affect the behavior of SCN juveniles (Warnke et al. 2008), the juveniles in extracts from leguminous crops, soybean, red clover, and sunn hemp had a significantly higher percentage of individuals that were paralyzed after 48 and 72 hours of exposure compared to non-leguminous crops oilseed rape, Illinois bundleflower, and corn.

Overview and Objectives

There is an increasing interest in using cover crops as a tool to help mitigate the SCN problem, but there are insufficient data to reach confident conclusions as to whether there is a real potential for cover crops to decrease SCN population densities. The data that have been published thus far were inconsistent, collected from poorly designed experiments, yielded no significant results, or had a combination of these shortcomings. There are companies that market specific cultivars of cover crop species for their ability to reduce, or even eradicate, SCN in the soil, but there are not sufficient data to support these claims. More research is needed that is designed in a very intentional manner in order to draw analogies to field conditions in order to make more relevant inferences about this question. 14

The objectives of this research were to examine if and how cover crops cultivars that are advertised as decreasing SCN population densities affect SCN populations in experiments on a wide range of scale, including farmer-conducted strip trials, small-plot field studies, and greenhouse and laboratory experiments.

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CHAPTER 2. DETERMINING THE IMPACTS OF COVER CROPS ON SOYBEAN CYST NEMATODE HATCHING, CHEMOTAXIS, AND ROOT PENETRATION

A paper to be submitted to the journal Plant Disease

Chelsea J. Harbach, Elizabeth Wleizen, and Gregory L. Tylka

Iowa State University, Department of Plant Pathology and Microbiology, 1344 Advanced Teaching and Research Building, Ames, IA 50011

Abstract

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 serving as trap crops. Experiments were conducted to determine if root exudates and soil leachates (RE/SL) from cover crop plants affected SCN hatching and chemotaxis and if there were significant differences in root penetration by SCN juveniles among the different cover crop plant types. Cover crops with potential to serve as trap crops may stimulate hatching and/or attract hatched SCN juveniles and be penetrated by large numbers of nematodes that cannot feed. In fourteen-day-long laboratory experiments, there was greater SCN hatching in crimson clover RE/SL than in RE/SL from all other cover crop treatments, although hatching was not consistently greater than in water. Hatching in RE/SL from rapeseed and mustard was less than in water. In laboratory experiments, SCN juveniles were not attracted or repelled to RE/SL from any cover crop treatment. And in twenty-day-long greenhouse experiments, more SCN juveniles penetrated the roots of crimson clover, mustard, and rapeseed than all other cover crops. Few SCN juveniles penetrated the roots of annual ryegrass and cereal rye. These results indicate 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 those investigated in these experiments. 21

Introduction

In United States soybean production, the soybean cyst nematode (SCN), Heterodera glycines Ichinohe, ranks consistently as the top yield-suppressing pathogen (Allen et al. 2017;

Koenning and Wrather 2010; Wrather and Koenning 2009). Methods to manage SCN typically include growing resistant soybean cultivars in rotation with nonhost crops and using nematode- protectant seed treatments (Niblack 2005; Bissonnette and Tylka 2017). The lack of genetic diversity in resistance available in commercial soybean cultivars has, over time, selected for

SCN populations that are able to reproduce on resistant soybean plants (Howland et al. 2018;

McCarville et al. 2017). Decreased effectiveness of soybean resistance to SCN has made continued management of the pest progressively more difficult.

The use of cover crops is an increasingly popular practice for farmers in the United States

(CTIC 2017). Cover crops alleviate soil erosion and reduce nutrient leaching (Clark 2007). They also may have an adverse effect on SCN population densities. Cover crops may reduce SCN population densities by serving as a trap crop, releasing nematicidal allelochemicals, producing inhibitory allelochemicals, and/or stimulating hatching (Niblack and Chen 2004). A cover crop would serve as a trap crop if a large number of nematode juveniles entered the crop roots and subsequently perished as a result of arrested development and starvation due to lack of feeding.

Trap crops have been effective at reducing population densities of several other plant- parasitic nematodes. Marigold species (Tagetes L. spp.) are well-established trap crops for

Meloidogyne incognita, the root-knot nematode (Hooks et al. 2010), and specific cultivars of oilseed radish (Raphanus sativus var. oleiferus) can act as a trap crops for Heterodera schachtii, the sugar beet cyst nematode (Hafez and Sundararaj 2009). Riggs (1987) found that SCN juveniles penetrated the roots of 27 different plant species, but juveniles did not enter the roots of 22

25 additional plant species. There were no data reported on how many nematodes were found in the roots of the plants from experiments conducted by Riggs (1987), and to our knowledge, there have been no published reports on whether cover crops can serve as trap crops for SCN.

There have been two published studies documenting the effects of cover crops on SCN hatching. Results of the studies were variable and somewhat inconclusive. Warnke (2008) found that after 14 days, significantly more SCN juveniles hatched in leachates of sand in which red clover (Trifolium pratense) and sunn hemp (Crotalaria juncea), both leguminous species, were grown compared to hatching in leachates from fallow (non-planted) sand and sand in which the non-cover crop, SCN nonhost corn (Zea mays) were grown. They also observed a significant suppression of hatching by leachates from soil in which a legume, Illinois bundleflower

(Desmanthus illinoensis), a crucifer, rapeseed (Brassica napus), and a grass, perennial ryegrass

(Lolium perenne), were grown compared to the controls. However, the treatment effects were not consistent among multiple runs of the experiments and hatching in a positive control treatment in the experiments (ZnCl2) was low relative to hatching in the water control. Studies with SCN conducted by Riga et al. (2001) used plant root exudates collected from cover crop treatments grown in perlite, and there was greater hatching in exudates from a grass, annual ryegrass

(Lolium multiflorum), and a legume, white clover (Trifolium repens), compared to the soybean control. Non-cover crop and positive hatch control treatments were not included in these experiments for comparison.

There are no published reports of how cover crops affect specific aspects of SCN biology and behavior other than hatching. Researchers have recently developed new methods to assess

SCN chemotaxis. Beeman et al. (2016) created microfluidic chips to observe SCN second-stage juvenile movement towards or away from compounds, including known attractants or repellants. 23

And Jensen et al. (2018) used these microfluidic chips to determine how the leachates from soybean seeds with various seed treatments planted in soil attracted or repelled SCN second- stage juveniles. Such experimental methods can be applied to determine how cover crops affect

SCN chemotaxis.

The objectives of this study were to determine if leachates of soil in which cover crops are grown affect hatching and chemotaxis of SCN juveniles and whether there are differences in how many juveniles penetrate the roots of cover crop plants

Materials and Methods

Collection of Root Exudates and Soil Leachates

The root exudate and soil leachate (RE/SL) collection method used was adapted from

Sikora and Noel (1996). Seeds of cover crops were sown in 600-cm3-capacity plastic Cone- tainers (Stuewe and Sons, Inc., Tangent, Oregon, USA) filled with a steam pasteurized mix of

1:1 construction sand and field soil (a Webster clay loam from Ames, Iowa, not infested with

SCN). There were three sets of experiments conducted using the following treatment groups: A) one representative cultivar of seven selected cover crop species, B) eight different broadleaf cover crop cultivars, and C) three cultivars of cereal rye and two of annual ryegrass (Table 1).

All of the cover crop cultivars selected for these experiments do not support SCN reproduction

(Kobayashi-Leonel et al. 2017). Each experiment also included a non-planted soil control and a non-cover crop, SCN nonhost tomato (Solanum lycopersicum cultivar Rutgers) control. One to two weeks after planting, plants in each Cone-tainer were thinned to a desired number, which varied by species (Table 1). Plants were watered every other day for 4 weeks and watered for the last time two days prior to collection of RE/SL.

The RE/SL were collected by dispensing 30 ml of sterile deionized (DI) water into each

Cone-tainer and waiting for one hour before adding an additional 100 ml of sterile DI water into each Cone-tainer suspended above a glass beaker. The RE/SL were collected in the beaker as they leached out of the bottom of each Cone-tainer. There were four replications per treatment, and all of the RE/SL were combined per treatment. If a replication did not contain enough plants to be within an acceptable range (as stated in Table 1), the replication was not included in the

RE/SL collection. Consequently, the RE/SL for each treatment varied in total volume but the total volume collected was always enough for subsequent use in experiments regardless of missing replications.

The RE/SL were filter sterilized using a Corning® 150 ml bottle-top filter with 0.22-µm pores (Corning, New York, USA) on 50-ml centrifuge tubes connected to a vacuum. The RE/SL were stored in 50-ml plastic centrifuge tubes (Thermo Fisher Scientific Inc., Waltham,

Massachusetts, USA) at 4ºC until they were used in hatching experiments and stored at -20ºC until used in chemotaxis experiments.

Hatching Experiments

Eggs of SCN for hatching experiments were obtained from the roots of the susceptible soybean cultivar Williams 82 grown in SCN-infested field soil (HG type 2) collected from

Muscatine, Iowa, for 4 to 8 weeks in a greenhouse maintained at 27ºC. The soybean roots in culture were carefully removed from the soil, rinsed, then placed on an 850-µm-pore sieve nested over a 250-µm-pore sieve. The SCN females on the roots were dislodged by spraying with a stream of water. The eggs were extracted by crushing the females on a 250-µm-pore sieve with a rubber stopper (Faghihi and Ferris 2000) and then collecting the eggs on a 25-µm-pore sieve nested under a 75-µm-pore sieve. The eggs were separated from any remaining soil debris 25 by sucrose centrifugation (Jenkins 1964) followed by rinsing thoroughly with tap water prior to use in hatching experiments.

The hatching experiments were conducted in sterile six-well plates (Corning® Corstar®,

Tewksbury, MA, USA), with one plate serving as a single experimental unit. In the top left well of each plate, 4 ml of treatment solution were deposited. The treatment solutions were RE/SL from the various cover crops, from the non-cover crop, SCN nonhost tomato, and from non- planted soil. Sterile DI water and a known SCN hatch stimulant, 5 mM zinc sulfate (ZnSO4)

(Tefft and Bone 1984), were included as control treatments. A sieve with 30-µm-pore nylon mesh (Elko Filtering Co., Switzerland) was placed into the well, and 200 to 400 SCN eggs were added into each sieve. The plates were incubated in the dark at 25ºC for three days, then the sieves with the unhatched eggs were moved to an adjacent well in the plate and fresh solution was added to the well. When SCN juveniles hatched in the sieves, they moved down through the mesh and were in the liquid within the well. This process of transferring sieves to wells with fresh solution was repeated on day seven. On the fourteenth day of the experiment, sieves were removed from the plates, and the number of hatched juveniles in each well plus the remaining unhatched eggs on each sieve were counted. The cumulative percent hatch was calculated by dividing the total number of juveniles that hatched after 14 days by the total number of hatched juveniles plus the remaining unhatched eggs. Subsequently, the proportion hatch for each treatment was calculated by dividing the cumulative percent hatch of the treatment by the cumulative percent hatch in the non-planted soil RE/SL control. Each experiment was organized using a randomized complete block design and was repeated once with four replications of each treatment per run of the experiment.

Chemotaxis Experiments

Microfluidic chemotaxis chips were fabricated out of polydimethylsiloxane (PDMS)

(DOW Sylgard® 182 Silicone Elastomer) using methods described by Beeman et al. (2016). The four lanes of each chip (Fig 1A) were filled with DI water to remove all air bubbles, then three lanes of a chemotaxis chip were randomly selected to receive a treatment opposite of a DI water control in the reservoirs. Both reservoirs of the remaining fourth lane were filled with DI water.

The lane with DI water in both reservoirs was a control treatment used to determine if the movement of the juveniles in the chip was biased towards one side of the chip or the other. Data from chemotaxis chips that had more than 10% difference in the number of nematodes observed in the left and right resting chambers of lanes containing DI water on both sides were not used in analyses. Treatments were randomly assigned chip lane (1-4) and side (left or right) locations.

Solutions of 0.5M KNO3 and 0.5M CaCl2 were used as attractant and repellant control solutions, respectively (Beeman et al. 2016). A total of 45 μl of one of the cover crop RE/SL treatments, one of the control solutions, or DI water were dispensed according to the treatment randomization. Eggs of SCN were extracted and collected as described for hatching experiments above. Second-stage juveniles of SCN were collected by placing eggs on a sieve with 30-µm- pore nylon mesh in petri dishes submerged in DI water, sealed with Parafilm® and incubated for

3 to 5 days at 25°C in the dark. Active, hatched juveniles (16 to 130) that passed through the nylon mesh were collected and dispensed into the center nematode entry port of each chemotaxis chip (Fig. 1). Chips were placed into a 10-cm-square petri dish, wrapped in Parafilm®, and incubated at 25°C in the dark for 24 hours.

After incubation, chips were observed at 25x magnification using a stereoscope, and the juveniles were counted and recorded as being in the resting chamber (Fig 1B) nearest the 27 treatment reservoir, the DI water reservoir, or remaining in the center nematode entry port of each lane. The number of juveniles in each of the three areas in the lane were divided by the total number of nematodes in the lane to calculate percent. There were four replications per treatment per experimental run, and the experiment was repeated two to three times, depending on the treatment.

Root Penetration Experiments

Mixtures of construction sand, field soil from Ames, Iowa (Webster clay loam), and soil naturally infested with SCN (from Fruitland, Iowa, HG type 2, identified as a Fruitfield coarse sand) were prepared for three runs of the experiment. All mixtures were a sandy loam texture.

The soil mixture in the first run was a 2:2:1 ratio of construction sand: field soil: SCN-infested soil. A 2:1:2 ratio mix of construction sand: field soil: SCN-infested soil was used for the remaining runs. The initial population densities for the first, second, and third runs of this experiment were approximately 2,500, 3,900, and 3,500 eggs/100 cm3, respectively. Cone- tainers with a 150-cm3-capacity (Stuewe and Sons, Inc., Tangent, OR, USA) were filled with the soil mixture and placed into 7.6-liter-capacity buckets filled with sand. A single replication of each treatment fit into one bucket, and thus, the experiment was set up using a randomized complete block design.

Seeds of multiple cover crop species, tomato (a non-cover crop, SCN nonhost), and an

SCN-susceptible soybean cultivar (Table 1) were sown in replicate Cone-tainers and watered every other day for 20 days in a growth chamber maintained at 25ºC with a 16-hour photoperiod.

Cone-tainers were thinned to one plant per Cone-tainer three to ten days after planting. However,

Cone-tainers with annual ryegrass and cereal rye often had more than one plant per experimental unit remaining because of difficulties in removing the growing point of these plants from of the 28 soil. After 20 days, the tops of plants were removed and discarded, and the roots were rinsed thoroughly to wash away as much residual soil debris as possible, then weighed to determine fresh root mass. Roots were placed into one well of a six-well plate (Corning® Corstar®,

Tewksbury, MA, USA) and stored at -20ºC prior to processing.

The nematodes were extracted from the plant roots using a method modified from Jensen et al. (2018) as follows. The day before processing, frozen plant roots were thawed to room temperature (ca. 21ºC) and then were frozen again at -20ºC overnight. On the day of processing, the roots were thawed at room temperature again, cut into approximately 1-cm-long pieces, and blended in 125 ml of tap water in a 350-ml-capacity Waring® blender (Torrington, Connecticut,

USA) by pulsing 10 times and then blending for 30 seconds, consecutively. After blending, the mixture was poured through an 850-µm-pore sieve over a 25-µm-pore sieve. Root debris were caught on the 850-µm-pore sieve, and the nematodes were recovered on the 25-µm-pore sieve.

The 850-µm-pore sieve was rinsed 10 times before collecting the material from the 25-µm-pore sieve to ensure that no nematodes remained on the top sieve. The materials on the 25-µm-pore sieve were collected, stained with acid fuchsin (Byrd et al. 1983), and observed under a dissecting microscope at 15x magnification. The total number of nematodes were counted for each experimental unit. The first and third runs of the experiment had six replications and run two had three replications.

Data Analyses

Data were analyzed in SAS 9.4 (SAS Institute Inc., Cary, NC, USA). Hatching experiment analyses were conducted on the natural-log-transformed data after calculating the cumulative proportion hatch for each treatment relative to the hatch in the non-planted RE/SL control for each replication in order to meet assumptions for the analysis of variance (ANOVA). 29

Subsequently, the proportion data for the non-planted soil RE/SL control treatment were excluded from the analysis as it was used to calculate the proportion and, consequently, always had a value of 1.0. An ANOVA was performed using SAS Proc MIXED for each experiment.

The standard error of the mean was calculated based on the average, the standard deviation, and sample size for each treatment. The hatch proportion means were further analyzed using SAS

Proc TTEST to determine if the values were significantly different from 1.0, e.g. different than the proportion of the non-planted RE/SL control. The means shown in Fig. 2 are backwards- transformed values.

Chemotaxis experiments were analyzed in two ways with two different response variables: (1) the percent of nematodes that were attracted and repelled from the RE/SL treatments and (2) the proportion of nematodes attracted and repelled from the RE/SL treatments relative to the non-planted soil RE/SL control. The first analysis, conducted on both response variables, was a pair of ANOVAs using SAS Proc MIXED to test for differences among treatments within a treatment group towards or away from RE/SL treatments. The standard error of the mean was calculated for each treatment as described above. The second analysis conducted the proportion response variable was a set of paired t-tests using SAS Proc TTEST to determine whether there was a difference in repulsion or attraction for each treatment compared a value of 1.00 as described above in the analyses of the hatching experiments.

The results from the root penetration experiments were analyzed using natural-log- transformed data of the total number of SCN juveniles per root system and the number of nematodes per fresh root gram in an ANOVA using Proc MIXED. The same formula was used to calculate the standard error of the mean for each treatment. The presented data are means and standard error of the means. 30

Results

Hatching Experiments

On average, hatching after 14 days in DI water ranged from 28 to 41 percent and was 71 to 75 percent in ZnSO4 among experiments with the different treatment groups (data not shown).

And hatching in RE/SL from the non-planted soil control treatment in the experiments was 15 to

23 percent (data not shown), which was consistently and usually significantly less than hatching in deionized water (Fig. 2). There was significantly greater hatching in RE/SL from crimson clover than in RE/SL from the non-planted soil control treatment (α = 0.05) in experiments with both treatment group A (Fig. 2A) and treatment group B (Fig. 2B). Hatching in the RE/SL of all other cover crop treatments and tomato was not significantly different than hatching in the non- planted soil control treatment (Fig. 2)

Chemotaxis Experiments

Juveniles of SCN were attracted to the KNO3 control treatment with an average of 33.0 percent of nematodes moving towards the compound versus 8.0 percent found in in the DI water resting chamber (data not shown). Juveniles were repelled by the CaCl2 control treatment with

22.8 percent of juveniles found in resting DI water resting chambers away from the treatment compared to 6.9 percent that moved towards the treatment (data not shown). On average, 16.7 percent of SCN juveniles were attracted to the non-planted soil RE/SL and 14.7 percent were repelled by the same treatment. The juvenile nematodes were neither attracted nor repelled by the RE/SL of the cover crop treatments in any experimental group (Fig. 3). The results from the analyses conducted on the percent of nematodes attracted and repelled from the RE/SL treatments were not different from the results from analyses conducted on the proportion of nematodes in a treatment compared to the non-planted soil RE/SL control (data not shown) 31

Root Penetration Experiments

There were large differences in the numbers of SCN juveniles that penetrated the roots of the cover crop plants in the experiments (Fig. 4). The total number of juveniles recovered from the roots of the susceptible soybean control treatment was significantly greater than all other treatments (Fig. 4A). When root mass was taken into consideration, crimson clover had a numerically larger amount of SCN juveniles found within the roots, although the mean was not significantly different from the susceptible soybean control (Fig. 4B). Crimson clover also had the second-highest number of nematodes in the roots after soybeans when total number of juveniles found per plant was analyzed (Fig. 4A). The number of SCN juveniles found in crimson clover roots was significantly greater than many of the other broadleaf and all grass cover crop plants in the experiments in both analyses (Fig. 4). The roots of all cultivars of cereal rye and annual ryegrass consistently had the fewest juvenile nematodes in them, and the numbers for those treatments always were significantly lower than for any other treatments in these experiments (Fig. 4).

Discussion

The cover crop treatment that had the most SCN juveniles in plant roots was also the treatment that stimulated SCN hatching, crimson clover (Trifolium incarnatum). These results may indicate that this cover crop has potential to serve as a trap crop for SCN. Crimson clover is one of several leguminous plant species used as a cover crop. Alfalfa (Medicago sativa) and red clover, both leguminous plant species, decreased SCN population densities when inter-seeded into a standing soybean crop (Chen et al. 2006). However, the reduction was not consistent throughout all years and locations of the experiment. Warnke et al. (2008) observed significantly more SCN hatching in leachates from two different leguminous species, but another leguminous 32 species included in the experiments inhibited hatching. More work with a variety of leguminous cover crops is warranted to determine if they affect SCN hatching. And more research is also needed to determine if there are consistent decreases in SCN population densities when crimson clover is grown in SCN-infested fields. Including other leguminous cover crops in future work would also be beneficial. However, in the United States few farmers use leguminous plant species as their sole cover crop. Leguminous plant species are usually included in a cover crop mix (CTIC 2017). The effects of cover crops species that we observed in greenhouse and laboratory studies may not occur under field conditions, particularly when the leguminous cover crop species are used in mixes.

The grass cover crop species and cultivars included in our experiments did not have high numbers of juveniles in plant roots or have effects on SCN hatching or chemotaxis. We did not take into account the possibility that different concentrations of the RE/SL in our experiments may affect the biology of SCN differently. The hatching of SCN (Tefft and Bone 1984) and specific behaviors (Schroeder and MacGuidwin 2010) are known to vary depending on concentration of the compounds being studied. Huisman (1982) explains how most root exudates are produced at root tips and not along the root zone of elongation. Taking this into consideration, it is likely that the concentration of root exudates in RE/SL collected from grass cover crops was greater than what was collected from broadleaf cover crop species. Future work with cover crops RE/SL and SCN would benefit from determining a way to take relative number of root tips into consideration when preparing concentrations for experimental use. The intention of our experiments was to collect RE/SL in a manner that would best mimic field conditions to identify the initial candidates could produce significant effects on SCN biology and behaviors. 33

The next steps of this process should involve looking at different concentration of cover crop

RE/SL in similar experiments.

Of all of the cover crop species studied in our experiments, crimson clover had the highest number of SCN juveniles penetrate the plant roots, but there were some cruciferous cover crops with similar numbers of nematodes per gram of root. This result may suggest that there is a potential for these cruciferous cover crops to serve as a trap crop for SCN as well.

Other studies have observed root penetration of cruciferous plants by SCN juveniles. Warnke et al. (2008) reported that SCN juveniles penetrated all cover crop species tested, including one cruciferous species, oilseed rape, but specific numbers of nematodes that entered the plant roots in these experiments were not reported. Also, Riggs (1987) determined that SCN juveniles penetrated the roots of the cruciferous plant, turnip (Brassica rapa), but no data were reported on how many. While we are not the first to report that SCN penetrated the roots of a cruciferous species, we did observe root penetration of more species than previously reported. And to our knowledge, ours is the first study to quantify SCN penetration in the roots of various crop species, revealing some evidence that there is potential for some cover crops to serve as trap crops for SCN. Furthermore, we know that the cover crops used in these studies are not hosts for

SCN reproduction, with no SCN females developing on brassica cover crops and few (less than five) found on the roots of leguminous cover crops (Kobayashi-Lenonel et al. 2017).

Another factor to consider while interpreting these results is the overall relative root mass of these different cover crop species in the soil. Researchers at Iowa State University assessed the root biomass for a standalone oat (Avena sativa L.) cover crop as well as a cover crop mix including oats, radish, and hairy vetch (Vicia villosa Roth) over three years (Licht et al. 2018).

On average, the hairy vetch root biomass made up less than 10 percent of the total root biomass 34 of the cover crop mix and radish roots made up less than 20 percent of the biomass. While it does look like crimson clover and perhaps other brassica species may have the most promise as a trap crop for SCN, the likelihood of having a sufficient amount of root biomass to encounter as many nematodes as possible may not be very high. More research is needed to determine how root biomass of trap crop candidates affects the actual ability of the cover crop to lower SCN population densities.

The results of our laboratory experiments indicated that the RE/SL of some cover crops affected SCN hatching but had no effect on SCN chemotaxis. There were also differences in root penetration by SCN among these cover crop species. But these cover crops must be tested in field experiments to determine if the effects are consistent on a larger scale. If certain cover crop species can decrease SCN population densities in infested fields, recommendations can be made to farmers looking for alternative methods to manage the growing SCN problem. Furthermore, as the use of cover crops continues to increase in the United States, having scientifically rigorous answers regarding their effects on other aspects of crop production, including plant diseases, will be highly valuable.

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Figure 1. Microfluidic chip (Beeman et al. 2016) used in chemotaxis experiments. One chip (A) consists of four parallel, independent lanes. Juveniles were dispensed into the central nematode entry port of each lane, and treatment or control solutions were dispensed into the outer reservoirs to allow diffusion of treatments through the filters into the resting chambers and eventually diffusing towards the nematode entry port. Juveniles could migrate towards and into either of the resting chambers (B) depending on how they reacted to the treatments.

Figure 2. The proportion of soybean cyst nematode (SCN) juveniles hatched in root exudate/soil leachate (RE/SL) as compared to hatch in the non-planted soil control for three hatching experiments (A) treatment group A (one species of each cover crop treatment), (B) treatment group B (broadleaf cover crops), and (C) treatment group C (annual ryegrass and cereal rye cultivars. The different cultivars are designated numerically following the name of the species and information on the specific species can be found in Table 1. Control treatments included deionized (DI) water and a known SCN hatch stimulant, ZnSO4. The standard error of the means is designated by the error bar for each proportion mean. Values significantly different than 1.0 (the non-planted soil control) are indicated by * P < 0.05 or ** P < 0.01. Data were averaged over two experimental runs with four replications each). 39

Figure 3. The proportion of soybean cyst nematode (SCN) juveniles that moved towards water and cover crop RE/SL compared to movement in the non-planted control for three sets of experiments (A) treatment group A (one species of each cover crop treatment included in this study), (B) treatment group B (broadleaf cover crops), and (C) treatment group C (cereal cover crop cultivars). The different cultivars are designated numerically following the name of the species and information on the specific species can be found in Table 1. Control treatments 2 included CaCl , known to repel SCN, and KNO3, a known SCN attractant. The standard error of the means is designated by the error bar for each proportion mean. An asterisk (*) indicates that the proportion mean was significantly different from the value of 1.0, or the non-planted soil RE/SL control (* = P < 0.05, ** = P < 0.01). 40

Figure 4 Total number of soybean cyst nematodes (SCN) in root systems (A) and the number of SCN per root gram (B) in the roots of cover crops, SCN-susceptible soybean, and non-cover crop, SCN nonhost tomato. The different cultivars are designated numerically following the name of the species and information on the specific species can be found in Table 1. The standard error of the means is designated by the error bar for each proportion mean. Plants are listed in descending order of number of nematodes from left to right. Data are from three experimental runs with six replications in runs 1 and 3 and three replications in run 2. 41

Table 1. Cover crop, non-cover crop/nonhost, and susceptible host control treatments used in experiments.

Treatment Species Cultivar Source Plants per Cone-tainera annual ryegrass 1 Lolium multiflorum Bounty Saddle Butte Ag., 10-12 Inc. Annual ryegrass 2 Lolium multiflorum RootMax Cover Crop 10-12 Solutions Annual ryegrass 3 Lolium multiflorum King LaCrosse Seed 10-12

Cereal rye 1 Secale cereale Aroostoock Public 3-5 Mustard 1 Brassica juncea Kodiak Mighty Mustard 5-8 Mustard 2 Brassica juncea Pacific Gold Mighty Mustard 5-8 Daikon radish 1 Raphanus sativus var. CCS779b Smith Seed 3-5 longipinnatus Services Oilseed radish 1 Raphanus sativus var. Image LaCrosse Seed 3-5 oleiferus Oilseed radish 2 Raphanus sativus var. Terranova Midwest Grass & 3-5 oleiferus Forage Cereal rye 3 + Secale cereale + SF 102 LaCrosse Seed 3-5, Crimson clover + Trifolium incarnatum + Cover 5-6, Daikon radish 3 Raphanus sativus var. Starter mixc 3-5 mix longipinnatus Annual ryegrass 1 + Lolium multiflorum + Soil Buster Saddle Butte Ag., 10-12, Daikon radish 2 Raphanus sativus var. mixd Inc. 3-5 mix longipinnatus Annual ryegrass 1 + Lolium multiflorum + Synergist Saddle Butte Ag., 10-12, Rapeseed mix Brassica napus mixd Inc. 3-5 Tomato Solanum lycopersicum Rutgers Public 1-2 Soybean Glycine max Williams 82 Public - a The number of plants per cone-tainer were determined based on the size of the plants. b CCS779 is the cultivar that was previously sold as Tillage® radish was trademarked by Cover Crop Solutions. The cultivar is now sold by Smith Seed Services under this new name. c There are no cultivar names specified for the species in this mix. d Cultivars in Soil Buster mix were Bounty annual ryegrass and Enricher radish. Cultivars in Synergist mix were Bounty annual ryegrass and Dwarf Essex rapeseed. 42

CHAPTER 3. ASSESSING THE DIRECT AND RESIDUAL EFFECTS OF COVER CROP GROWTH AND WINTERKILLED COVER CROPS ON SOYBEAN CYST NEMATODE POPULATION DENSITIES

Modified from a manuscript to be submitted to the journal Plant Disease

Chelsea J. Harbach and Gregory L. Tylka

Iowa State University, Department of Plant Pathology and Microbiology, 1344 Advanced Teaching and Research Building, Ames, IA 50011

Abstract

The use of cover crops has been increasing in the United States, but their effects on soybean cyst nematode (SCN), Heterodera glycines, are not well established. Greenhouse experiments were conducted to determine if cover crops directly decrease SCN population densities or have residual effects on SCN reproduction. After 60 days of growth in SCN-infested soil, SCN population densities had not significantly decreased compared to the non-planted soil control for any of the nine cover crop treatments or the three cover crop mixes. Susceptible soybean plants were grown in soil remaining from this experiment, and fewer SCN females formed on soybean roots following annual ryegrass 1, annual ryegrass 2, annual ryegrass 3, daikon radish, mustard 1, mix 1, and the tomato control compared to following the non-planted control. In another set of experiments, cover crops were grown for 56 days and winterkilled by a

28-day exposure to Iowa winter conditions. The population density of SCN was not significantly decreased for any cover crop treatment compared to the non-planted soil control. There were fewer SCN females that formed on soybean plants grown following seven of the cover crop treatments, including all three annual ryegrass treatments and the tomato control, compared to soybeans grown following the non-planted soil control. In summary, there were no significant 43 reductions of SCN population densities and an adverse residual effect of cover crops on SCN reproduction may have occurred.

Introduction

The soybean cyst nematode (Heterodera glycines Ichinohe), SCN, has been considered the top yield-suppressing pathogen of United States soybean (Glycines max L.) for nearly two decades (Allen et al. 2017; Koenning and Wrather 2010; Wrather and Koenning 2009).

Managing SCN typically involves growing a rotation of nonhost crops and SCN-resistant soybean cultivars. However, seed companies have largely relied on a single source of SCN resistance for control. In Iowa, approximately 95% of the commercial soybean cultivars possess

SCN resistance genes from PI88788 (Tylka and Mullaney 2018). The prolonged use of a single source of resistance genes is causing a shift in SCN populations towards increased reproduction on resistant cultivars (Howland et al. 2018; McCarville et al. 2017). Subsequently, SCN is becoming increasingly difficult to manage, and new and innovative management tools are needed.

Traditional crop production systems are becoming more environmentally conscious. One example of this trend is the use of cover crops. Over the past decade, the land area planted to cover crops has steadily increased throughout the United States (CTIC 2017). Cover crops are primarily grown to mitigate soil erosion and reduce nutrient leaching (Clark 2007) and to increase in soil organic matter (CTIC 2014). Little is known about the impact of cover crops on plant pathogens, such as SCN.

An important consideration in growing cover crops is whether they serve as suitable hosts for SCN reproduction. Growing cover crops that are hosts for SCN would result in an unintentional increase in the population density of the pathogen. Riggs (1987) conducted 44 experiments to determine and observe is SCN penetrate and continue to develop in the roots of

50 different plant species with multiple cultivars of some of the species. There were 25 plant types that had no SCN penetration, 22 species that SCN penetrated but did not develop, 7 plants that SCN penetrated and only slightly developed, and 5 plant species that SCN developed to maturity on. However, few of the plants used in this study are candidates for use in cover cropping. One recent study in Iowa showed that SCN does not reproduce on most plants that are typically grown as cover crops, with the exception of some leguminous plants on which there were low amounts of reproduction, not exceeding five females per plant (Kobayashi-Leonel et al.

2017). Thus, it appears unlikely that most cover crops will serve as inadvertent hosts supporting a significant amount of SCN reproduction under field conditions.

There is great interest in the possibility that cover crops could reduce SCN population densities, perhaps partially because of reported successes of their use in reducing population densities of other plant-parasitic nematodes. In one instance, researchers found that some cover crops, especially oilseed radishes (Raphanus sativus var. oleiferus), significantly reduced the nematode population densities in fields infested with sugar beet cyst nematode, Heterodera schachtii (Hafez and Sundararaj 2009). As H. schachtii is a close relative of SCN (Potter and

Fox 1965), it is possible that some cover crops may reduce SCN population densities as well.

A few published field and greenhouse experiments reported overall decreases in SCN population densities in soil in which various cover crops were grown, but the results were inconsistent among experiments (Wen et al. 2017; Chen et al. 2006; Warnke et al. 2008; Warnke et al. 2006; Pedersen and Rodriguez-Kabana 1991). The most recent study in Illinois found lower

SCN population densities in soil samples collected in the spring following rapeseed (Brassica napus), cereal rye (Secale cereale), and canola (Brassica napus) cover crops compared to the 45 non-planted control, but the lower SCN population densities occurred infrequently and inconsistently over years and locations (Wen et al. 2017). Furthermore, there were no initial soil samples collected from the plots in the experiments to use to determine actual changes in SCN population densities during the time that the cover crops were grown. And another study reported that SCN population densities in soil in which cover crops were grown were not significantly different from the densities in the plots that had no cover crops planted (Miller et al. 2006).

Few greenhouse studies have examined interactions of cover crops and SCN. Warnke et al. (2006) conducted experiments with 46 different crop treatments and found no significant decreases in SCN population densities for any cover crop treatment compared to the non-planted soil control. Thus far, results of studies investigating the interaction between SCN and cover crops in field and greenhouse conditions have been inconsistent or not significant.

The objectives of this study were to examine (i) if cover crops reduce SCN population densities, (ii) if there is a residual effect of cover crops on SCN reproduction on soybeans grown following cover crops, and (iii) if winterkilled cover crops affect SCN population densities and the subsequent effect on SCN reproduction on soybean in greenhouse experiments.

Material and Methods

Experiments to Assess Direct Effects of Cover Crops on SCN Population Densities

The growth medium used in this experiment was a mixture of construction sand, field soil

(a non-infested Webster clay loam from Ames, Iowa), and naturally SCN-infested soil from

Muscatine, Iowa, with a sandy loam texture. The naturally infested soil was mixed with sand and non-infested field soil to achieve a target initial SCN population density of 5,000 eggs/100 cm3.

The first run of this experiment used a mixture of a 3:2:5 ratio of construction sand: field soil:

SCN-infested soil and the second run used a 1:2:7 ratio. The average initial population densities 46 in these experiments were 5,490 eggs/100 cm3 and 5,338 eggs/100 cm3 for runs 1 and 2, respectively.

An experimental unit consisted of one 600-cm3-capacity Cone-tainer (Stuewe and Sons,

Inc., Tangent, OR, USA) filled with the SCN-infested soil mixture. For each experimental unit, a subsample of at least 100 cm3 of the soil mixture that was used to fill the Cone-tainer was collected to determine the initial SCN population density (Pi). Cone-tainers filled with the soil mixture were placed into 19-liter-capacity plastic boxes filled with construction sand. One box held one replication of all treatments, so the experiment was setup in a randomized complete block design (RCBD). Seeds of cover crops, an SCN-susceptible soybean cultivar, and a non- cover crop, SCN nonhost tomato (Table 1) were planted in the Cone-tainers, and one Cone-tainer per replication was left non-planted as an additional control. The plants were thinned to a desired number of plants per Cone-tainer (Table 1), determined by considering the plant size and

“ground cover” for each species. The first run of this experiment was conducted in a greenhouse water bath maintained at 27ºC for 60 days from June to September 2016. The second run was conducted for 60 days from July to September 2017 in a growth chamber with daytime and nighttime temperatures of 27ºC and 18ºC, respectively, and a 12-hour photoperiod to simulate the environment of Iowa in early fall. Sixty days after planting, the aboveground plant tissues were cut and discarded, the soil mixture with chopped root tissue from each Cone-tainer was mixed individually. After mixing, the root tissue from each experimental unit was removed to the best of our ability prior to collecting a subsample of at least 100 cm3 to determine the final SCN population density (Pf).

To determine SCN population densities, cysts were extracted from the 100 cm3 subsamples of the soil mixture using a wet-sieving extraction method modified from Gerdemann 47

(1955). The cysts were collected on a 250-µm-pore sieve and were stored at 4ºC until processing was completed. To extract eggs, cysts were placed on a 250-µm-pore sieve and crushed using a mechanized rubber stopper (Faghihi and Ferris 2000). The eggs were collected on a 25-µm-pore sieve nested under a 75-µm-pore sieve (Faghihi and Ferris 2000). Extracted eggs were stained with acid fuchsin (Niblack et al. 1993) to improve visualization for counting. Population change factors (PCF) were calculated for each experimental unit by calculating the value of Pf/Pi (Miller et al. 2006). Analysis was conducted on natural-log-transformed PCF, after the PCF were calculated with the non-transformed Pi and Pf values for each experimental unit. The data from the soybean control were not included in the analysis because the PCF were very large as the soybean plants were a favorable host for SCN reproduction.

Experiments to Assess the Residual Effects of Cover Crops on SCN Reproduction

After the experiments described above concluded, a 150-cm3 subsample of the soil mixture from each experimental unit was collected and transferred to a 150-cm3-capacity Cone- tainer into which three SCN-susceptible soybean seeds (cultivar Williams 82) were sown. The

Cone-tainers were thinned to one soybean plant each after seedling emergence. The Cone-tainers were placed into 7.6-liter-capacity buckets filled with construction sand in an RCBD where each bucket was equivalent to one block. The Cone-tainers were incubated in a water bath maintained at 27ºC in a greenhouse room with a 16-hour photoperiod.

After 30 days, soybean plants were removed individually from the Cone-tainers. The excess soil mixture was carefully removed from the roots by soaking in tap water, and then SCN females were dislodged from the roots with a stream of water while the roots lay on an 850-µm- pore-sieve nested over a 250-µm-pore sieve. The SCN females gathered on the 250-µm-pore 48 sieve were stored prior to counting. The fresh root weight was recorded for each plant after SCN females were removed. The SCN females were counted under a dissecting microscope.

Because SCN-susceptible soybean plants were grown as a treatment for 60 days in the previous experiment, the Pf for the soil mixture from these experimental units was very large, and the soybean roots of plants grown in the soil mixture were severely stunted. Thus, the soybean treatment was removed from the dataset for the analyses of this experiment. This experiment was analyzed using the square-root-transformed data of the total number of SCN females formed per plant.

Experiments to Assess the Effect of Winterkilled Cover Crops on SCN Population Densities

The soil mixture used for this experiment was a mixture of construction sand, field soil, and naturally infested soil from Muscatine, Iowa, as described above. The soil mixture for both experimental runs was a 1:2:7 ratio of construction sand: field soil: SCN-infested soil. Run 1 had an average initial SCN population density of 7,354 eggs/100 cm3 and run 2 averaged 6,795 eggs/100 cm3. The experimental units were 15 × 15 cm plastic pots 20 cm deep with a volume of

4.7 L (Sunlight Supply Inc., Vancouver, Washington, USA). A subsample of at least 100 cm3 of the soil mixture was taken from each experimental unit after pots were filled, prior to seeding, and used to determine the Pi. Seeds of cover crop treatments, tomato (the non-cover crop, SCN nonhost), and an SCN-susceptible soybean cultivar (Table 1) were sown in experimental units, and one pot in each replication was left non-planted as a control.

The pots were organized as an RCBD in a growth chamber with a 12-hour photoperiod and daytime/nighttime temperatures of 27ºC/18ºC to simulate the environment in late summer/early fall in Iowa, the ideal time for cover crop seeding. Pots were watered every three days. The two runs of the experiment were planted in early December 2016 and late November 49

2018. Following 56 days of plant growth, the pots were moved outside to the freezing winter environment in Iowa. The average high and low temperatures in February 2017 during experimental run 1 were 2ºC and -4ºC, respectively. And in February 2019, when experimental run 2 was placed outside, the average high and low temperatures were -7ºC and -15ºC, respectively. After 28 days unsheltered outside, the pots were brought inside. The soil mixture and dead roots from each experimental unit (or in the case of the non-planted control, just the soil mixture) were emptied into a plastic bin and mixed thoroughly. The root debris was removed to the best of our ability. A subsample of at least 100 cm3 was collected from the soil mixture to determine the Pf for each experimental unit. The calculated PCF was natural-log-transformed for analysis, as previously described.

An additional 150-cm3 subsample of the soil mixture from each pot was collected and placed into a 150-cm3-capacity Cone-tainer, and the SCN-susceptible soybean cultivar Williams

82 was grown for 30 days. At the end of this bioassay, SCN females were removed from soybean roots and counted as previously described. Analyses were performed on the square-root- transformed data of the total number of SCN females formed per plant.

Data Analyses

Data for all experiments were subjected to analysis in SAS 9.4 (SAS Institute, Cary, NC).

The data transformations were done to improve the normality of the distribution of the residuals in order to meet analysis of variance (ANOVA) assumptions. Experiments were performed twice. The repeated runs for these experiments were combined for analyses after determining that there were no significant differences between runs. Data were analyzed in an ANOVA using

Proc MIXED. Additionally, all PCF means were analyzed using Proc TTEST to determine if the values were significantly different from the average of the non-planted control. The soybean data 50 were not included in any analyses due to the final population density of the soil mixture from the

SCN-susceptible soybean treatment being extremely high, but the means for results from the soybean control are included in the figures. The reported data include the mean and standard error of the mean for each treatment.

The data from the SCN bioassays from both experiments were analyzed similarly using an ANOVA in using SAS Proc Mixed. These data were also subject to a series of paired t-tests to determine if the mean number of SCN females formed per soybean plant grown soil in which cover crops were previously grown was significantly different from the number of SCN females that formed on soybean plants grown following the non-planted soil control. The data presented are mean values and standard error of the means for each treatment.

Results

Direct Effects of Cover Crops on SCN Population Densities in Greenhouse Conditions

The SCN population densities decreased numerically over the 60-day duration of the experiment for all cover crop treatments and for the tomato and non-planted controls, with PCF ranging from 0.41 to 0.81 (Fig. 1A). There was no significant treatment effect on the change in

SCN population densities (PCF) among all of the cover crop treatments, tomato, and the non- planted soil control when analyzed with an ANOVA. The PCF for the soybean control (not included in the analysis) indicated a fourteen-fold increase in SCN population density. When t- tests were performed to determine if individual PCF values were significantly different from the non-planted soil control, there were no significant differences detected (a = 0.05).

Residual Effects of Cover Crops on SCN Population Densities in Greenhouse Conditions

The minimum number of SCN females recommended to form on an SCN-susceptible soybean cultivar in order to obtain valid results from greenhouse experiments is 100 (Niblack et 51 al. 2002; Niblack et al. 2009). There were 130 to 308 SCN females that developed on soybean roots grown in the soil mixtures following the growth of the cover crop treatments as well as the tomato and non-planted controls (Fig. 1B). There were significantly fewer SCN females found on the roots of soybean plants grown following six cover crop treatments and the tomato control compared to the number of SCN females that developed on soybean roots grown following the non-planted soil control (a = 0.05) (Fig. 1B).

Direct and Residual Effects of Winterkilled Cover Crops on SCN Population Density

After a 56-day growing period followed by 28 days of exposure to winter conditions, the

PCF among the cover crop treatments ranged from 0.76 to 1.05 (Fig. 2A). There were no significant differences in PCF detected among all cover crop treatments or the non-planted control when analyzed by an ANOVA (P > 0.05) (Fig. 2A). There were no significances observed in the series of paired t-tests conducted to determine if PCF of cover crop treatments were different from the non-planted soil control (Fig. 2A).

The total number of SCN females formed on susceptible soybean plants grown in the soil mixtures following the period of cover crop growth and winter exposure ranged from 516 to 812 in this experiment (Fig. 2B). There were 733 females formed on soybean roots following the tomato treatment and 931 females following non-planted soil. Significantly fewer (a = 0.05)

SCN females formed on soybean plants following six cover crop treatments and the tomato control compared to following the non-planted soil control (Fig. 2B

Discussion

In the 60-day long experiment the SCN population densities decreased numerically for all cover crop treatments, however there were no significant decreases in SCN population densities detected compared to the non-planted soil control. Meanwhile, the decreases in SCN population 52 density were less evident or nonexistent in the 84-day experiment although there were no differences among treatments. Warnke et al. (2006) similarly observed no significant differences among cover crop treatments and the non-planted control in decreasing SCN population densities in a greenhouse study. The difference in scale of the detected population density reductions between experiments could have be a function of differences in sampling error related to the size of the experimental units. The volume of the pots used in the winterkilled cover crop experiment was nearly eight times greater than the volume of the Cone-tainers used in the 60-day experiment. The variability of SCN population densities within and among small-plot field experiments has been demonstrated by Pérez-Hernández and Giesler (2017). It is possible that differences in sampling error associated with using containers of different sizes in the two types of greenhouse experiments may have affected our ability to detect differences on SCN population densities among treatments to the same extent in the experiments.

Interestingly, there appeared to be no significant decrease in SCN population densities as a result of growing Mix 1 in either experiment. One of the components of Mix 1, crimson clover

(Trifolium pretense L.), has exhibited some characteristics of a good SCN trap crop candidate.

Crimson clover root exudates and soil leachates positively influence SCN hatching and researchers found large numbers of SCN juveniles in the plant roots (Harbach et al. 2019). Based on this information, it was unexpected that Mix 1 did not decrease SCN population densities more. This may be partially explained by the ratio of root mass that was actually crimson clover in this mix compared to the other species components. Licht et al. (2018) found that the average biomass of hairy vetch (Vicia villosa Roth) roots made up less than 10 percent of the total root biomass of a cover crop mix in field experiments consisting of oat (Avena sativa L.), radish, and hairy vetch. This may translate into our greenhouse experiments with a similar cover crop mix. 53

Furthermore, it is possible that the use of a crimson clover as a standalone cover crop could have a higher potential to decrease SCN population densities based on these findings. More work is needed to determine if these effects of crimson clover, and possibly other legumes, can be observed in field conditions. However, the use of leguminous species as standalone cover crops is not common, rather these species are generally included as a components of cover crop mixes

(CTIC 2017).

There were significantly fewer SCN females that developed on soybean plants grown following annual ryegrass 1, annual ryegrass 2, and annual ryegrass 3, cereal rye 1, mustard 1, daikon radish, mix 1, mix 2, and mix 3 compared to the non-planted soil control across both experiments. This result suggests the potential for negative residual effects of cover crops on the reproduction of SCN. Riga et al. (2001) reported fewer SCN in soil surrounding soybean roots following incorporation of cover crop residues from some treatments (eight different leguminous species, annual ryegrass, and perennial ryegrass). Also, Warnke et al. (2006) reported significantly fewer SCN females on soybean roots grown in soil following three leguminous plant species compared to a fallow control. In our study, there were some soybean plants grown following cover crop treatments that had similar numbers of SCN females as the non-planted control. So, it appears that not all cover crop species or cultivars have a negative residual effect on SCN reproduction. While the observation of residual effects is not a novel discovery, we found this effect following more cover crops than previously reported, including multiple annual ryegrass cultivars, brassica species, cover crop mixes, and the non-cover crop, SCN nonhost control tomato.

In summary, based on the way we measured effects in our greenhouse experiments, there were no cover crop treatments that significantly decreased SCN population densities compared to 54 the non-planted control. There were some negative residual effects on SCN reproduction under the conditions of our experiments. More studies are necessary to assess the impacts of cover crops on SCN population densities in field systems, especially to determine if there are any decreases by cover crop that are greater than that that is seen by simply rotating with a nonhost crop.

Acknowledgements

We thank the United States Department of Agriculture North Central Sustainable

Agriculture Research and Education graduate student grant program for help in funding this research. Thanks to Greg Gebhart for collecting the SCN-infested soil from Fruitland, Iowa, used in these studies. And thanks also to Corey Tjaden for his help with processing the soil samples from these experiments.

Literature Cited

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Hafez, S. L. and Sundararaj, P. 2009. Evaluation of trap crops for Heterodera schachtii management in sugar beet production. J. Nematol. 41:335.

Harbach, C. J., Wlezien, E., and Tylka, G. L. 2019. Determining the impacts of cover crops on soybean cyst nematode hatching, chemotaxis, and root penetration. In prep.

Howard, D. D., Chambers, A. Y., and Lessman, G. M. 1998. Rotation and fertilization effects on corn and soybean yields and soybean cyst nematode populations in a no-tillage system. Agron. J. 90:518-522.

Koenning, S. R. and Wrather, J. A. 2010. Suppression of soybean yield potential in the continental United States by plant disease from 2006 to 2009. Plant Health Prog. doi:10.1094/PHP-2010-1122-01-RS

Niblack, T. L., Heinz, R. D., Smith, G. S., and Donald, P. A. 1993. Distribution, density, and diversity of Heterodera glycines in Missouri. J. Nematol. 25:880-886.

Niblack, T. L., Arelli, P. R., Noel, G. R., Opperman, C. H., Orf, J. H., Schmitt, D. P., Shannon, J. G., Tylka, G. L. 2002. A revised classification scheme for genetically diverse populations of Heterodera glycines. J. Nematol. 34: 279-288.

Niblack, T., Tylka, G. L., Arelli, P., Bond, J., Diers, B., Donald, P., Faghihi, J., Ferris, V. R., Gallo, K., Heinz, R. D., Lopez-Nicora, H., Von Qualen, R., Welacky, T., and Wilcox, J. 2009. A standard greenhouse method for assessing soybean cyst nematode resistance in soybean: SCE08 (standardized cyst evaluation 2008). Online. Plant Health Progress doi:10.1094/PHP-2009-0513- 01-RV.

Pedersen, J. F., and Rodriguez-Kabana, R. 1991. Winter grass cover crop effects on nematodes and yields of double cropped soybean. Plant Soil. 131:287-291.

Pérez-Hernández, O. and Giesler, L. J. 2017. Intra- and interplot variability of Heterodera glycines population densities in experimental settings to soybean variety evaluations in Nebraska. Crop Protection 94: 123-136.

Potter, J. W. and Fox J. A. 1965. Hybridization of Heterodera schachtii and H. glycines. Phytopathology 55: 800-801.

Riggs, R. D. 1987. Nonhost root penetration by soybean cyst nematode. J. Nematol. 19:251-254.

Schroeder, N. E. and MacGuidwin, A. E. 2010. Mortality and behavior in Heterodera glycines juveniles following exposure to isothiocyanate compounds. J. Nematol. 42:194-200.

Tylka, G. L., and Mullaney, M. P. 2018. Soybean cyst nematode-resistant soybeans for Iowa. Iowa State University Extension and Outreach Publications, no. 1649. https://lib.dr.iastate.edu/extension_pubs/100

Warnke, S. A., Chen, S., Wyse, D. L., Johnson, G. A., and Porter, P. M. 2008. Effect of rotation crops on hatch, viability and development of Heterodera glycines. Nematology 10:869-882.

Wen, L., Lee-Marzano, S., Ortiz-Ribbing, L. M., Gruver, J., Hartman, G. L., and Eastburn, D. M. 2017. Suppression of soilborne diseases of soybean with cover crops. Plant Dis. 101:1918-1928. 57

Wrather, A. and Koenning, S. 2009. Effects of diseases on soybean yields in the United States 1996 to 2007. Plant Health Prog. doi:10.1094/PHP-2009-0401-01-RS 58

Table 1. Cover crop, non-cover crop/nonhost, and susceptible host control treatments used in greenhouse experiments.

Abbreviation Treatment Species Cultivar Source Plants per Cone- tainera ARG 1 annual ryegrass Lolium multiflorum Bounty Saddle Butte 10-12 Ag., Inc. ARG 2 annual ryegrass Lolium multiflorum RootMax Cover Crop 10-12 Solutions ARG 4 annual ryegrass Lolium multiflorum King LaCrosse Seed 10-12 CR 1 cereal rye Secale cereale Aroostoock Public 3-5 M 1 mustard Brassica juncea Kodiak Mighty Mustard 5-8 M 2 mustard Brassica juncea Pacific Mighty Mustard 5-8 Gold DR daikon radish Raphanus sativus var. CCS779a Smith Seed 3-5 longipinnatus Services OSR 1 oilseed radish Raphanus sativus var. Image LaCrosse Seed 3-5 oleiferus OSR 2 oilseed radish Raphanus sativus var. Terranova Midwest Grass 3-5 oleiferus & Forage Mix 1 cereal rye + Secale cereale + SF 102 LaCrosse Seed 10-12 crimson clover + Trifolium incarnatum + Cover daikon radish Raphanus sativus var. Starter mixb mix longipinnatus

Mix 2 annual ryegrass + Lolium multiflorum + Soil Buster Saddle Butte 10-12 daikon radish Raphanus sativus var. mixc Ag., Inc. mix longipinnatus Mix 3 annual ryegrass + Lolium multiflorum + Synergist Saddle Butte 10-12 rapeseed mix Brassica napus mixc Ag., Inc. T tomato Solanum lycopersicum Rutgers Public 1-2 S soybean Glycine max Williams 82 Public 1 a The number of plants per cone-tainer was determined by the plant size and “ground cover”. b CCS779 was previously sold as Tillage® radish by Cover Crop Solutions. The cultivar is now sold by Smith Seed Services but is no longer named Tillage radish. c There are no cultivars listed for the species in this mix. d Cultivars in Soil Buster mix were Bounty annual ryegrass and Enricher radish. Cultivars in Synergist mix were Bounty annual ryegrass and Dwarf Essex rapeseed. 59

Figure 1. The (A) average population change factor (PCF) of soybean cyst nematode (SCN) after 60 days of cover crop growth and the (B) mean number of SCN females formed on soybean plants grown in soil for 30 days following 60 days of cover crop growth with associated standard error of the means for each treatment. (A) The PCF were calculated as the ratio of the final SCN population density to the initial population density for each treatment. Values less than 1.0 indicate a decrease in SCN population density. (B) The treatments that had significantly fewer SCN females form per soybean root following cover crop treatments compared to the non- planted (NP) soil control are indicated by * P < 0.05 or ** P < 0.01. These data were collected over two experimental runs with six replications each. Cover crop abbreviations and cultivar specifications are found in Table 1. 60

Figure 2. The (A) average population change factor (PCF) of soybean cyst nematode (SCN) after 56 days of cover crop growth followed by winterkilling the plants with 28 days of exposure to winter weather in Iowa, and the (B) mean number of SCN females formed on soybean plants grown in soil for 30 days following after the cover crops were winterkilled, with associated standard error of the mean for each treatment. (A) The PCF were calculated as the ratio of the final SCN population density to the initial population density for each treatment. Values less than 1.0 indicate a decrease in SCN population density. (B) The treatments that had significantly fewer SCN females form per soybean root following winterkilled cover crops compared to the non-planted (NP) soil control are indicated by * P < 0.05 or ** P < 0.01. These data were collected over two experimental runs with six replications each. Cover crop abbreviations and cultivar specifications are found in Table 1. 61

CHAPTER 4. THE EFFECTS OF COVER CROPS ON SOYBEAN CYST NEMATODE POPULATION DENSITIES IN MULTI-YEAR, SMALL-PLOT FIELD EXPERIMENTS IN IOWA

A paper to be submitted to the journal Plant Disease

Chelsea J. Harbach and Gregory L. Tylka

Iowa State University, Department of Plant Pathology and Microbiology, 1344 Advanced Teaching and Research Building, Ames, IA 50011

Abstract

The impacts of cover crops on soybean cyst nematode (SCN), Heterodera glycines, population densities were studied in small-plot experiments conducted in 2016-2017 and 2017-

2018 at the Iowa State University Muscatine Island Research Farm (MIRF) and Northern

Research Farm (NRF). There were two experiments at each farm distinguished by the crop initiating a corn-soybean rotation in 2016. Cover crops were seeded in late summer annually.

Soil samples were collected to determine SCN population densities when cover crops were seeded, again in late fall, and a third time in spring before corn or soybeans were planted.

Population change factors (PCF) were calculated to determine changes in population densities between sampling intervals. There was no year, location, or field combination in which there were significant differences among PCF for cover crop treatments and the non-planted control.

Furthermore, there were no significant differences detected between treatment PCF means and the mean of the non-planted plot control and there were no consistent changes in SCN population densities for any single treatment across fields or in any experiment at either farm. The variability is PCF throughout these experiments is likely a function of the natural spatial variability of SCN and possibly the differences in soil texture between the NRF and MIRF 62 experiments. Overall, none of the cover crops consistently decreased SCN population densities under the conditions of these experiments.

Introduction

The soybean cyst nematode (SCN), Heterodera glycines (Ichinohe), has been the top yield-suppressing pathogen of soybean (Glycines max L.) in the United States for the past two decades (Allen et al. 2017; Koenning and Wrather 2010; Wrather and Koenning 2009). For many years, farmers were able to manage SCN by growing a nonhost crop, such as corn (Zea mays), in rotation with SCN-resistant soybean cultivars. Since the 1990s, the resistant soybean market has been dominated by soybean cultivars with SCN resistance derived from PI 88788 (McCarville et al. 2017). And research results now indicate the increased ability of SCN populations to reproduce on soybean cultivars with PI 88788 source of resistance (Howland et al. 2018;

McCarville et al. 2017). Even as some farmers in the Midwest are using longer rotations with multiple years of corn between single years of soybean, the long-term survivability of SCN in the soil makes the probability of eradicating the pest unlikely (Niblack 2005).

Farmers are looking for additional and alternative approaches to mitigate SCN. One practice that is of increasing interest coincides with the increasing concerns for the environmental and ecological impacts of large-scale, row-crop agriculture. In Iowa, cover crops are sown into fields either late in the season into a standing crop or shortly after crop harvest to provide plant cover to fields that would otherwise be bare for nearly six months of the year. The land area planted to cover crops each year in the United States has steadily increased over the past decade (CTIC 2017). Known environmental benefits to growing cover crops include reducing nutrient leaching and mitigating soil erosion (Clark 2007), and most farmers report observing an overall improvement in soil health from using cover crops (CTIC 2017). 63

Previously published and unpublished information has piqued interest among farmers, agronomists, and seed dealers about whether cover crops can have an additional benefit of reducing SCN population densities. For example, a study published in 1988 reported that the

SCN population density after five years of continuous SCN-resistant soybean with no cover crop was greater than the same continuous SCN-resistant cultivar grown with an annual ryegrass

(Lolium multiflorum) cover crop (Dabney et al. 1988). Unpublished data from the University of

Illinois suggest that using Aroostook cereal rye (Secale cereale) or Bounty annual ryegrass as a cover crop can greatly reduce SCN population densities, or even eradicate the nematode, compared to a fallow control (Plumer unpublished, personal communication). Some studies have shown inconsistent effects of cover crops on SCN population densities. In a relatively recent study published from the University of Illinois, rapeseed (Brassica napus), cereal rye, and canola were found to decrease SCN population densities in a few locations, but not consistently (Wen et a. 2017). In contrast, Pedersen and Rodriguiez-Kabana (1991) found no significant differences in

SCN population densities when three different grass species, cereal rye, annual ryegrass, or wheat (Triticum aestivum), were grown after soybean in Alabama.

It is difficult to draw broad or robust conclusions on the effects of cover crops on SCN population densities based on the current published research. The purpose of this study was to determine whether various cover crop species can affect SCN population densities in Iowa.

Materials and Methods

Small-plot experiments were conducted at the Iowa State University (ISU) Muscatine

Island Research Farm (MIRF) in Fruitland, Iowa, and the ISU Northern Research Farm (NRF) in

Kanawha, Iowa, from the late summer of 2016 to the spring of 2018. There were two experiments at each farm distinguished by the crop starting the rotation in 2016, either corn- 64 soybean (C-S) or soybean-corn (S-C). The soils in the fields in which the S-C and C-S experiments were conducted at the MIRF were Fruitfield coarse sand with 92% sand, 2.5% silt,

5% clay and 80% sand, 10% silt, and 10% clay, respectively. The average initial SCN population densities at the time of cover crop seeding were 9,803 eggs/100 cm3 for the S-C experiment and

578 eggs/100 cm3 for the C-S experiment (Table 1). Soil in which the C-S experiment at the

NRF was conducted was Canisteo clay, with 30% sand, 32% silt, 37% clay with an average initial population density of 813 eggs/100 cm3, and the soil in which the S-C experiment was conducted was a Clarion loam with 42.5% sand, 30% silt, and 27.5% clay and an average initial population density of 2,131 eggs/100 cm3 (Table 1). An SCN-susceptible cultivar, Pioneer

93M11, was grown in the soybean year of all rotations in experiments at both farms. The corn hybrids used in the experiments varied by year and location (Table 1). Weeds were managed using glyphosate in addition to conventional pre- and post-emergence herbicides.

The corn and soybean crops were bulk seeded into the fields, and 0.9-m alleys were cut into the fields to delineate the plots after the corn and soybean crops became established. Each experiment consisted of 60 four-row plots with 0.75-m row spacing, measuring 3 meters wide by

5.2 meters long. In late summer of each year (late August to early September), seeds of nine different cover crop treatments (Table 1) were sown into the standing corn and soybean plots using a hand-crank fertilizer spreader (Earthway Products Inc., Bristol, Indiana, USA). In mid to late October (Table 1), the corn and soybean crops were bulk harvested, and the grain was removed from the plots. No grain yield data were collected for these experiments. There were ten treatments in total, including a non-planted control (Table 2). The seeding rates used for each cover crop treatment were taken from Clark 2007. The cover crop treatments included two cultivars each of annual ryegrass, cereal rye, and mustard (Brassica juncea), one cultivar of 65 oilseed radish (Raphanus sativus var. oleiferus) and daikon radish (Raphanus sativus var. longipinnatus), and one cover crop mix (cereal rye, daikon radish, and crimson clover [Trifolium incarnatum]) (Table 2). Each treatment was replicated six times per experiment, organized in a randomized complete block design.

The cover crop treatments were applied to the same exact plot area over the multiple years of the experiments. Fields received no tillage to avoid disruption of the SCN population density spatial patterns over time. The corn and soybean crops were planted as close to the previously planted corn and soybean rows as possible in order to reduce variability in the sampling area over time. After seeding the cover crops at the MIRF each year, the plots were irrigated with 12.7 mm of supplemental water over a three-day period.

To assess the effects of cover crops on SCN population densities over time, five 2.5-cm- diameter soil cores were collected at a depth of 15 to 20 cm from each of the center two corn or soybean rows in every plot at the time of cover crop seeding. A second set of soil samples were similarly taken from the center two rows of each plot after harvest of corn and soybean in late

November prior to a hard frost. These samples were referred to as the pre-winter samples. The third soil sample collection date was after the plots were sprayed with glyphosate in the spring to terminate the cover crops, but prior to planting of corn and soybean

Soil Processing

Soil samples were dried at room temperature, around 22ºC, for 24 to 48 hours. After drying, the soil samples were crushed using a soil grinder (Humboldt Mfg. Co., Elgin, Illinois,

USA) with the screen removed. Cysts were extracted from a 100-cm3 subsample of each soil sample using an automated wet-sieving extraction method modified from Gerdemann (1955).

Extracted cysts were crushed using a mechanized rubber stopper to release the eggs (Faghihi and 66

Ferris 2000). The SCN eggs were stained using acid fuchsin (Niblack et al. 1993) and counted to estimate the population density for each subsample.

To determine the effects of cover crops on SCN population densities over time, two population change factors (PCF) were calculated for each plot. The PCF were ratios of population densities as described by Miller et al. (2006). The PCF1 was the pre-winter population density divided by the population density at the time of cover crop seeding, and the

PCF2 was the population density in the spring divided by pre-winter population density. The

PCF were calculated using raw SCN egg count data.

Data Analyses

An analysis of variance was conducted for each year, location, field, and PCF combination. Data were natural-log-transformed in order to meet assumptions of the analysis of variance. The data were analyzed using Proc MIXED in SAS 9.4 (SAS Institute, Inc. Cary, NC).

Additionally, each mean was subjected to a t-test using SAS Proc TTEST to determine if the

PCF for the cover crop treatments were significantly different than the PCF of the non-planted control. The presented PCF means, and standard error of the means were calculated conventionally.

Results

There was no significant effect of treatment in altering the SCN population density between sampling dates among the various treatments as revealed by the analysis of variance

(data not shown). The PCF for the period between seeding of the cover crop and winter (PCF1,

Figs. 1 and 2) ranged from 0.42 to 4.11 overall. And for the overwinter period (PCF2, Figs 1 and

2), the PCF ranged from 0.41 to 6.30. Overall, PCF were greater in the experiments conducted in 67 southern Iowa at the MIRF (Fig. 2) than those in the experiments at the NRF (Fig. 1). But the ranges in PCF were somewhat similar and overlapped.

There were no significant differences (P > 0.05) detected in the t-tests assessing whether the PCF in cover crop plots were larger or smaller than the PCF from the non-planted soil plots

(Fig. 1 and 2). The standard error of the mean for PCF were largely variable, ranging from 0.11 to 6.26. Of all 160 treatment means over two locations and two years and all PCF, 58 percent of the forty smallest standard error of the mean estimates occurred in treatments from the MIRF experiments (data not shown). Of the forty largest standard error of the means for PCF in these studies, 65 percent occurred in the NRF experiments (data not shown).

Discussion

There were no significant decreases in SCN population densities for any cover crop treatment compared to the non-planted control over two years, two locations, two rotations, and two sampling intervals in our experiments. While the PCF for some treatments were greater than

1.00, they were not statistically different from the PCF in the non-planted control within the same year, location, field, and sampling interval. Furthermore, any numerical increases in SCN population density are not likely due to SCN reproduction on the cover crops in the plots as research has shown there to be little to no development of SCN females on the roots of the cover crops used in these experiments (Kobayashi-Leonel et al. 2017). Knowing this, it is unlikely that the significant increases in SCN population densities between sampling periods has to do with

SCN reproduction on the cover crops. Rather, the observed SCN population density increases likely were due to sampling error associated with the natural variability in the distribution of

SCN throughout a field and even within a plot (Pérez-Hernández and Giesler 2017). 68

It is notable that of all treatment means over two years, two locations, and two fields, more than half of the 40 smallest standard error of the means were found in treatments at the

MIRF location, and 65 percent of the 40 largest standard error of the means were from treatments in fields at the NRF. The soil textures are quite different between locations and may affect the efficiency of extracting cysts used to determine the population density of the samples collected in these experiments. The soil in the MRF fields have a high sand content, and when these samples are placed into water prior to the automated wet sieving extraction process, any soil clods quickly dissociate (Tylka, personal communication). This means that the likelihood of extracting most or all of the cysts from the soil samples from the MIRF is very high. Conversely, the soil in fields from the NRF have a much higher loam content. Although the soils samples go through a soil grinder, small clods of soil can remain. When these samples are dumped in the water prior to cyst extraction, the clods have a lower tendency to dissociate (Tylka, personal communication). Thus, the odds of cysts occasionally being trapped in soil clods and skewing the population density estimate for soil samples collected from the NRF are much higher and could consequently impact the standard error of the means for these samples.

One of the components of the cover crop mix used in these experiments was crimson clover (Trifolium pratense), a leguminous species. A previous study has shown that crimson clover has the highest potential to serve as a trap crop for soybean cyst nematode compared all other cover crop species used in the experiments including annual ryegrass (Lolium multiflorum), cereal rye (Secale cereale), mustard (Brassica juncea), rapeseed (Brassica napus), and radish

(Raphanus sativus) (Harbach et al. 2019). This was determined by observing a greater number of

SCN juveniles in the roots of and a greater hatching rate in root exudates and soil leachates collected from crimson clover. However, our results from the field studies did not find a great 69 impact of the cover crop mix in reducing SCN population densities. This could be contributed to sampling variability, as previously described. Or, it may be partially explained by the overall difference in total root mass observed from leguminous species grown in a cover crop mix with a grass species and radish. Licht et al. (2018) observed that hairy vetch (Vicia villosa Roth), when grown in a mix with oats (Avena sativa L.) and radish, on average made up just 10 percent of the root mass from the cover crop mix. Furthermore, while crimson clover has exhibited some characteristics similar to that of a good trap crop, it is also unlikely that crimson clover would ever be grown as a standalone cover crop species. It is more common for leguminous species to be integrated as a component of a cover crop mix (CTIC 2017).

The success of cover crop establishment in the plots was estimated based on visual observation, but more in-depth, systematic data collection on cover crop stand was not performed. In general, cover crop establishment following soybeans was better than following corn for all of the treatments, although there were satisfactory stands of the cover crops in all plots in all fields. The annual ryegrass and cereal rye treatments consistently survived overwinter in all field experiments and had the most visible biomass on the soil surface in the spring. There was little evidence of biomass from brassica species in the spring, as they winterkilled each year and their plant residue degraded quickly. Oilseed radish are known to winterkill when exposed to consistent temperatures at or below -5ºC, and mustard usually dies with the first hard frost

(Clark, 2007). Our results detected no consistent change in SCN population densities as a result of any one cover crop treatment regardless of cover crop establishment and survival.

The results from our study are in contrast to Wen et al. (2017) who reported significant reductions of SCN egg population densities associated with rapeseed (3 of 10 site-years), cereal rye (3 of 12 site-years), and canola (2 of 6 site-years). Not every cover crop was evaluated in 70 every year at every location in their study and they used a single soil sampling date to assess the

SCN population density in the spring following the cover crop with no initial soil sample to determine the population density chage over time. Furthermore, the SCN population densities in some of the fields in which their experiments were conducted were overall quite low, with average observed SCN population densities over all site-years of 353 eggs per 100 cm3. A low initial SCN population density would likely make detecting differences among treatments difficult due to a smaller margin for error. Three out of eight site-years in our experiments had average initial SCN population densities that were less than 1,000 eggs per 100 cm3. However, by growing an SCN-susceptible soybean cultivar, the initial SCN population densities in these fields dramatically increased in subsequent site-years. By having higher SCN population densities in our experiments and three soil sampling dates comprising two sampling intervals per year, our experiments seemingly would have been more likely to detect effects of cover crops on

SCN population densities.

Aroostook cereal rye and Bounty annual ryegrass were reported to significantly reduce

SCN population densities, or even eradicate the nematode, compared to an unplanted control

(Plumer unpublished, personal communication). We did not observe similar effects of these two cover crop cultivars in our experiments. Previous reports of decreases in SCN population densities with Aroostook cereal rye and Bounty annual ryegrass were based on data from one soil sampling date collected in the spring following the cover crop and there were no initial SCN population densities with which to determine the changes in SCN population densities. In our experiments, there were multiple sampling dates and intervals over multiple years that provided a more detailed assessment of the effects of Aroostook cereal rye and Bounty annual ryegrass on

SCN population densities. 71

Overall, cover crops did not have any consistent, significant effects on the SCN population densities over the time intervals included in these experiments. The cover crops used in this study are not likely to reliably reduce SCN population densities in typical corn-soybean rotations in the Midwest and should not be used solely for SCN control. Rather, cover crops should continue to be implemented primarily for the known benefits that they provide to farmers.

Acknowledgements

We thank the United States Department of Agriculture North Central Sustainable

Agriculture Research and Education graduate student grant program for help in funding this research. We also thank Chris Marett, Mark Mullaney, EB Wlezien, Jefferson Barizon, and numerous undergraduate students for help with sampling and processing soil samples, as well as staff at the MIRF and NRF for maintenance of the small-plot experiments.

Literature Cited

Clark, A. (ed.). 2007. Managing Cover Crops Profitably, 3rd ed. Sustainable Agriculture Network, Beltsville, MD.

Dabney, S. M., McGawley, E. C., Boethel, D. J., and Berger, D. A. 1988. Short-term crop rotation systems for soybean production. Agron. J. 80:197-204.

Faghihi, J., and Ferris, J. M. 2000. An efficient new device to release eggs from Heterodera glycines. J. Nematol. 32:411-413.

Gavassoni, W. L., Tylka, G. L., and Munkvold, G. P. 2001. Relationships between tillage and spatial patterns of Heterodera glycines. Phytopathology 91:534-545.

Gerdemann, J. W. 1955. Relation of a large soil-borne spore to phycomycetous mycorrhizal infections. Mycologia 47:619-632.

Harbach, C. J., Wlezien, E., and Tylka, G. L. 2019. Determining the impacts of cover crops on soybean cyst nematode hatching, chemotaxis, and root penetration. In prep.

Koenning, S. R. and Wrather, J. A. 2010. Suppression of soybean yield potential in the continental United States by plant disease from 2006 to 2009. Plant Health Prog. Online. doi:10.1094/PHP-2010- 1122-01-RS.

Niblack, T.L., Heinz, R.D., Smith, G.S., and Donald, P.A. 1993. Distribution, density, and diversity of Heterodera glycines in Missouri. J. Nematol. 25:880-886.

Niblack, T. L. 2005. Soybean cyst nematode management reconsidered. Plant Dis. 89: 1020- 1026.

Pedersen, J. F. and Rodriguez-Kabana, R. 1991. Winter grass cover crop effects on nematodes and yields of double cropped soybean. Plant Soil. 131:287-291.

Tylka, G. L. and Flynn, P. 1999. Interpreting SCN soil sample results. Iowa State University Extension and Outreach Publications, no. IPM 61. https://lib.dr.iastate.edu/extension_pubs/101/

Wen, L., Lee-Marzano, S., Ortiz-Ribbing, L. M., Gruver, J., Hartman, G. L., and Eastburn, D. M. 2017. Suppression of soilborne diseases of soybean with cover crops. Plant Dis. 101:1918-1928.

Winstead, N. N., Skotland, C. B., and Sasser, J. N. 1955. Soybean cyst nematode in North Carolina. Plant Dis. Rep. 39:9-11.

Wrather, A. and Koenning, S. 2009. Effects of diseases on soybean yields in the United States 1996 to 2007. Plant Health Prog. Online. doi:10.1094/PHP-2009-0401-01-RS

Table 1. Field information and planting, cover crop seeding, harvest, cover crop termination, and soybean cyst nematode (SCN) soil sampling dates for experiments at the Iowa State University (ISU) Northern Research Farm (NRF) in Kanawha, Iowa, and the ISU Muscatine Island Research Farm (MIRF) in Fruitland, Iowa. There were two fields per farm, designated by the crop in the rotation, including corn-soybean (C-S), and soybean-corn (S-C).

Seeding Cover crop Spring Crop and rate seeding and Harvest Pre-winter Cover crop soil SCN a Location Field planting date (seeds/ha) soil sample date soil sample termination sample Pi1 2016-2017 NRF C-S Cornb, 23 Apr 86,446 31 Aug 14 Oct 17 Nov 5 May 12 May 813 S-C Soybean, 6 May 358,302 31 Augc 18 Oct 17 Nov 5 May 12 May 2,132 MIRF C-S Corn, 24 Apr 87,969 16 Sept 17 Oct 29 Nov 17 Apr 3 May 578 S-C Soybean, 13 May 271,816 16 Sept 14 Oct 29 Nov 17 Apr 3 May 9,803 2017-2018 NRF C-S Soybean, 1 Jun 383,013 29 Aug 24 Oct 17 Nov 17 May 30 May 2,827 S-C Corn, 9 May 85,251 29 Aug 18 Oct 17 Nov 17 May 30 May 887 74

MIRF C-S Soybean, 22 May 252,047 6 Sept 18 Oct 15 Nov 25 Aprd 16 May 6,848 S-C Corn, 28 Apr 84,016 6 Sept 24 Oct 15 Nov 25 Aprd 16 May 2,560 a The Pi1 was the average initial SCN population density for that field determined by the soil samples collected at cover crop seeding. The pre-winter soil sample estimated the Pf1 (final population density) to determine the population change factor (PCF) 1 for this sampling interval by dividing Pf1 by Pi1. The pre-winter soil sample also served as Pi2 and the spring soil sample taken estimated the Pf2. The PCF2 was calculated by dividing Pf2 by Pi2 to estimate the population change for this second sampling interval. b The corn hybrids planted at the NRF in 2016 and 2017 were Dekalb 54-38 and Pioneer 0157AMX, respectively. The corn hybrid used at the MIRF was Pioneer 1197AM both years. The SCN-susceptible soybean cultivar Pioneer 93M11 was used in all soybean plantings. c Most cover crops were seeded at this location on this date, but Image oilseed radish, SF102 Cover Starter cover crop mix, and Aroostook cereal rye were seeded on September 6. d The plots in which the cover crop plants had winterkilled initially were not sprayed with herbicide on the date of cover crop termination listed in the table. But all plots were sprayed with herbicide approximately 10 days after the initial herbicide application

Table 2. The cover crop species and cultivars included in experiments. Seeding rates were obtained from Clark (2007).

Treatment Seeding (abbreviation) Species Cultivar Company rate (kg/ha) mustard (M 1) Brassica juncea Kodiak Mighty Mustard 16.8 mustard (M 2) Brassica juncea Pacific Gold Mighty Mustard 16.8 annual ryegrass Lolium multiflorum Bounty Saddle Butte 33.6 (ARG 1) Ag., Inc. annual ryegrass Lolium multiflorum RootMax Cover Crop 33.6 (ARG 2) Solutions daikon radish Raphanus sativus var. CCS779a Smith Seed 15.7 (DR) longipinnatus Services oilseed radish Raphanus sativus var. Image LaCrosse Seeds 15.7 (OSR) oleiferus cereal rye (CR 1) Secale cereale Aroostook Public 179.3 cereal rye (CR 2) Secale cereale Guardian LaCrosse Seeds 179.3 cereal rye Secale cereal + SF102 Cover LaCrosse Seeds 67.3 crimson clover Trifolium incarnatum + Starter mixb daikon radish Raphanus sativus var. (Mix) longipinnatus a CCS779 was previously sold as Tillage® radish by Cover Crop Solutions. The cultivar is now sold by Smith Seed Services as CCS779. b There are no cultivars listed for the species in this mix.

Figure 1. Soybean cyst nematode (SCN) population change factors (PCF) for two small plot experiments conducted at the Iowa State University Northern Research farm from 2016 to 2018. The two experiments were designated by the grain crop rotation starting in 2016, either soybean- corn (A) or corn-soybean (B). The SCN population densities were monitored over time by collecting soil samples three times each year: at cover crop seeding (late August), pre-winter (mid-November), and spring (May). Two PCF were calculated each year: PCF1 = pre-winter SCN population density ÷ SCN population density at cover crop seeding and PCF2 = spring SCN population density ÷ pre-winter SCN population density. Values greater than or less than 1.0 indicate an overall increase or decrease in SCN population density for that time interval, respectively. Data presented are the average PCF and standard error of the mean for each mean estimate.

Figure 2. Soybean cyst nematode (SCN) population change factors (PCF) for two small plot experiments conducted at the Iowa State University Muscatine Island Research Farm from 2016 to 2018. The two experiments were designated by the grain crop rotation starting in 2016, either soybean-corn (A) or corn-soybean (B). The SCN population densities were monitored over time by collecting soil samples three times each year: at cover crop seeding (early September), pre- winter (mid-November), and spring (May). Two PCF were calculated each year: PCF1 = pre- winter SCN population density ÷ SCN population density at cover crop seeding and PCF2 = spring SCN population density ÷ pre-winter SCN population density. Values greater than or less than 1.0 indicate an overall increase or decrease in SCN population density for that time interval, respectively. Data presented are the average PCF and standard error of the mean for each mean estimate.

CHAPTER 5. GENERAL CONCLUSION

The goal of this research was to determine if and how cover crops affect soybean cyst nematode (SCN), Heterodera glycines, population densities. This was done by performing experiments on a wide range of scale from laboratory-based assays to small-plot field experiments. The laboratory and greenhouse experiments gave us an idea of what we might expect to see in field experiments. In the first research chapter (Chapter 2), we observed some significant effects of cover crop root exudates and soil leachates on SCN hatching and differences in the number of nematodes found in the cover crop roots. However, significant effects among treatments were not observed in applied experiments in Chapters 3 and 4, as the sampling error for SCN population densities increased along with sampling area in our experiments, from 600-cm3 Cone-tainers, to 4.7-liter pots, up to 15.6-m2 small plots.

The cover crop treatment that was most interesting in the laboratory studies was crimson clover (Trifolium incarnatum), a legume. Even though farmers seldom use leguminous species as standalone treatments when planting cover crops in their fields, the effects of crimson clover on

SCN population densities should be investigated further. In fact, the effects of more leguminous cover crops on SCN population densities should be investigated. If crimson clover holds promise as an SCN trap crop, it is likely that other leguminous species may have similar effects. It could be interesting and useful to investigate the effects of more cover crop mixes that include leguminous species on SCN population densities since legumes are more likely to be used as a component of a cover crop mix.

There is a clear disadvantage to increasing the size of experimental units when monitoring SCN population density changes, such as in our field experiments in Chapter 4. The more soil there is to sample from, the higher the sampling error becomes as SCN cysts are

79 aggregated in the soil and the eggs are aggregated within the cysts. Perhaps future field experiments working with cover crops and SCN should start in fields that do not have a history of SCN and be infested with SCN eggs prior to initiation of the experiments. This may help reduce the variability of SCN population densities among and within treatment plots as well as increase the likelihood of detecting significant treatment effects.

Since we observed no consistent significant decreases in SCN population densities as a result of the cover crop treatments, it is unlikely that the cover crops used in our experiments will reliably decrease SCN population densities in the Midwest. Rather, farmers are better off to continue implementing crop rotations including SCN nonhost, corn (Zea maydis), and SCN- resistant soybean cultivars with nematode-protectant seed treatments and stick to using cover crops primarily for their known environmental and ecological benefits. There are still many environmental benefits associated with using cover crops and there is little concern that cover crops will inadvertently increase SCN population densities.

APPENDIX: DETERMINING IF HIGH OR LOW SOYBEAN CYST NEMATODE POPULATION DENSITY LEVELS IMPACT ABILITY TO OBSERVE DIFFERENCES IN POPULATION CHANGE FACTOR DUE TO COVER CROPS

Introduction

Greenhouse experiments with soybean cyst nematode (SCN), Heterodera glycines, can assess a variety of response variables. Of particular interest regarding cover crops is whether a period of cover crop growth can significantly decrease the SCN population density compared to an unplanted control. It is possible that having greater initial SCN population densities may make detecting significant decreases among treatments more likely. The goal of the following preliminary experiment was to test the hypothesis that the initial SCN population density of soil influences the SCN population change factor (PCF) over a period of cover crop growth.

Materials and Methods

The growth medium used in this experiment was a mixture of construction sand, field soil

(a Webster clay loam from Ames, IA), and naturally SCN-infested soil from Muscatine, IA, with a sandy loam texture. The naturally infested soil was mixed with sand and non-infested field soil in two batches to obtain two target initial SCN population densities of 1,000 eggs/100cm3 (low) and 5,000 eggs/100cm3 (high).

The soil mixtures were used to fill 600-cm3-capacity Cone-tainers (Stuewe and Sons,

Inc., Tangent, OR, USA), with each Cone-tainer serving as a single experimental unit. A 100- cm3 subsample from the soil mixture used to fill each Cone-tainer was collected to determine the initial SCN population density (Pi).

After the Cone-tainers were filled, they were placed into 19-liter-capacity plastic boxes filled with construction sand. One box held one replication of all treatment combinations, resulting in the experiment being a randomized complete block design (RCBD). Cover crop

81 treatments included cereal rye (Secale cereal) cv. Aroostook, annual ryegrass (Lolium multiflorum) cv. Bounty, mustard (Brassica juncea) cv. Kodiak, daikon radish (Raphanus sativus var. longipinnatus) cv. CCS779, and oilseed radish (Raphanus sativus var. oleiferus) cv. Image.

Cover crop seeds, an SCN-susceptible soybean (Glycine max) cv. Williams 82, and a non-cover crop, SCN nonhost tomato (Solanum lycopersicum) cv. Rutgers were sown into the Cone-tainers, and one Cone-tainer per population density treatment was left non-planted as an additional control.

Cone-tainers were placed into a growth chamber with daytime and nighttime temperatures of 27ºC and 18ºC, respectively. The Cone-tainers were watered every three days for

60 days. After 60 days, the aboveground plant tissue was cut and discarded, the soil mixture with chopped root tissues for each experimental unit from each Cone-tainer were mixed separately, and a final sample of at least 100 cm3 was collected to determine the final SCN population density (Pf).

The SCN cysts were extracted from 100 cm3 samples of the soil mixtures using a wet- sieving extraction method modified from Gerdemann (1955). The cysts were collected on a 250-

µm-pore sieve and were subsequently crushed on a separate 250-µm-pore sieve using a mechanized rubber stopper (Faghihi and Ferris 2000) to extract the eggs from within the cysts.

The eggs were collected on a 25-µm-pore sieve and stained with acid fuchsin (Niblack et al.

1993) to aid with visualization for counting. The population change factor (PCF) was calculated for each experimental unit by taking the ratio of the Pf/Pi (Miller et al. 2006).

An analysis of variance was conducted using SAS Proc MIXED (SAS Institute, Cary, NC, USA) on the natural-log-transformed data after calculating the PCF value. Data from the soybean control were not included in the analysis as the PCF values were very large due to the soybeans

82 being a host. Thus, the analysis conducted was a 2 x 7 factorial in an RCBD. The reported least square means are backwards transformed and the raw mean PCF values for the soybean treatments are also included for reference. Subsequent t-tests were performed using SAS prof

TTEST to determine if PCF means were significantly different from a value of 1.00, which would indicate a significant increase (PCF > 1.00) or decrease (PCF < 1.00) in SCN population density.

Results

The population densities in the soybean treatment had a twenty-fold increase (Table 1).

Most cover crop treatments decreased the SCN population densities over the duration of the experiment, with PCF ranging from 0.52 to 1.36. There was no significant difference in PCF among all cover crop and SCN population density treatment combinations (P > 0.05) (Table 1).

None of the cover crop treatments or the non-planted soil or tomato controls had PCF that were significantly different from 1.00 (a = 0.05) (Table 1).

Discussion

There was no observed advantage of having a higher initial SCN population density in detecting differences the effects of decreasing SCN population density over time among cover crop treatments in this experiment. It is unlikely that experiments to observe the SCN PCF over time would benefit from having extremely high initial SCN population densities. For the purposes of experiments included in this dissertation, we will use that using a target initial population density of 5,000 eggs/100cm3 to test for significant differences among treatments.

Literature Cited

Faghihi, J., and Ferris, J. M. 2000. An efficient new device to release eggs from Heterodera glycines. J. Nematol. 32:411-413.

Gerdemann, J. W. 1955. Relation of a large soil-borne spore to phycomycetous mycorrhizal infections. Mycologia 47:619-632.

Niblack, T. L., Heinz, R. D., Smith, G. S., and Donald, P. A. 1993. Distribution, density, and diversity of Heterodera glycines in Missouri. J. Nematol. 25:880-886.

Table 1. Population change factors (PCF) for soybean cyst nematode following a growth period of cover crop treatments in a high-SCN-population-density soil mixture (approximately 5,000 eggs per 100cm3) and a low-SCN-population-density soil mixture (approximately 1,000 eggs per 100cm3). This experiment was conducted once with six replications.

PCF PCF PCFu t- (high (low Cultivar Species (total) testv density) t-test density) t-test Bounty Lolium multiflorum 0.96 ns 0.72 ns 1.28 ns

Aroostoock Secale cerale 0.52 ns 0.48 ns 0.55 ns Kodaik Brassica juncea 0.67 ns 0.67 ns 0.68 ns

CCS 779 y Raphanus sativus 1.05 ns 0.81 ns 1.36 ns var. longipinnatus

Image Raphanus sativus 0.74 ns 0.71 ns 0.78 ns var. oleiferus

Rutgersz Solanum 0.92 ns 1.10 ns 0.79 ns lycopersicum non-planted - 1.22 ns 0.94 ns 1.57 ns Williams 82 Glycines max 23.95 ** 19.60 ** 28.31 **

u PCF (population change factor) = final population density ÷ initial population density. Presented values are backwards transformed. v Results from t-tests estimating if a PCF is significantly different from the value of 1.0, indicating an increase (PCF > 1.00) or decrease (PCF < 1.00) in SCN population density. Significance of the t-test designated by ** = P < 0.01. y CCS779 was previously sold as Tillage® radish by Cover Crop Solutions. The cultivar is now sold as CCS779 by Smith Seed Services. z Tomato cultivar Rutgers served as a non-cover crop, SCN nonhost control for this experiment.