Potato Nematode Research: with Special Reference to Potato-Early Die, Corky Ringspot and Soil Enzymes

POTATO NEMATODE RESEARCH: WITH SPECIAL REFERENCE TO POTATO-EARLY DIE, CORKY RINGSPOT AND SOIL ENZYMES

Loren George Wernette

A Thesis

Submitted to Michigan State University In partial fulfillment of the requirements For the degree of

Masters of Science

Entomology

2011 Abstract

POTATO NEMATODE RESEARCH: WITH SPECIAL REFERENCE TO POTATO-EARLY DIE, CORKY RINGSPOT AND SOIL ENZYMES

Loren George Wernette

Potato production faces numerous challenges three of these include Potato-Early Die

(PED), Corky Ringspot Disease of Potato (CRSD), and soil quality issues. This thesis consists of three chapters: 1) Impact of Alfalfa on Soil-Borne Enzymes in Potato Systems. 2) Vertical

Distribution of Paratrichodorus pachydermus, Pratylenchus penetrans (Nematoda), and

Verticillium dahliae (Mycota) in Michigan Potato Systems. 3) Soil Fumigation Guide. Soil quality was evaluated comparing soil enzyme activities associated with fields that had been in alfalfa for ten years in two year potato rotations with corn. We found that the alfalfa rotation had higher enzyme activities for phosphatase and tyrosine aminopeptidase. We also found that the mineralization rate of carbon was significantly higher in the alfalfa system, compared to the potato rotation. The nematode pathogens for CRSD and PED were evaluated to determine the depth at which the highest population density of Pratylenchus penetrans, Paratrichodorus pachydermus, and Verticillium dahliae were found in a potato system. We found that the majority of P. pachydermus was found below, a 30 cm soil depth in September. We also found that P. penetrans was found most commonly in the upper 30 cm of the soil. When targeting the virus vector, P. pachydermus. Fumigant and non-fumigant nematicides were tested. As the amount of active ingredient of the chemicals increased, however, the percent of CRSD symptom expression in the tubers decreased. We also showed that there was no increase in symptom expression during tubers storage.

Dedication

I would like to dedicate this thesis to my beautiful fiancé Meghan who has loved and supported me throughout this process.

iii

Acknowledgements

I would like to thank my major professor Dr. George W. Bird for all his time and guidance I could not have completed this research without this help. I would also like to thank my committee members

Dr. William Kirk and Dr. Chris Dizonzo for all of the help they have given me during the research and writing process. I would also like to thank Dr. Stuart Grandy, and Kyle Wickings and their lab for assisting with soil enzymes and carbon mineralization analysis. I would not have been able to accomplish what I have without the help of John Davenport, Rob Schafer, and Chris Long. They helped me with everything from chemical applications harvesting and storage sampling as well as just being good friends.

Without Walther’s Farms, especially KarI Richie, I would not have had the field sites to do a good portion of my research. I would like to thank Mark Otto for his guidance and understanding as I progressed through this process. I look forward to working with you for many years.

Table of Contents

List of Tables……………………………………………………………………………………………………………………..……vi.

List of Figures…………………………………………………………………………………………………………….………….viii.

Chapter I. Impact of Alfalfa on Soil-Borne Enzymes in Potato Systems……………………………………….1 1. Introduction……………………………………………………………………………………………………………..2 2. Methods…………………………………………………………………………………………………………………..5 3. Results……………………………………………………………………………………………………………………10 4. Discussion………………………………………………………………………………………………………………12 5. Conclusions…………………………………………………………………………………………………………….19

Chapter II. Vertical Distribution of Paratrichodorus pachydermus, Pratylenchus penetrans (Nematoda), and Verticillium dahliae (Mycota) in Michigan Potato Systems……………………………21 1. Abstract………………………………………………………………………………………………………………….21 2. Introduction……………………………………………………………………………………………………………22 3. Methods…………………………………………………………………………………………………………………26 4. Results……………………………………………………………………………………………………………………29 5. Discussion………………………………………………………………………………………………………………30 6. Conclusions…………………………………………………………………………………………………………….33

Chapter III. Corky Ringspot Disease of Potato Fumigant and Non-fumigant Nematicide control…………………………………………………………………………………………………………………..…………………35 a. Introduction………………………………………………………………………………………………..35 b. Methods……………………………………………………………………………………………………..36 c. Results………………………………………………………………………………………………………..39 d. Discussion……………………………………………………………………………………………………40 e. Conclusions…………………………………………………………………………………………………42

APPENDIX. Fumigation Field guide…………………………………………………………………………………………64

References……………………………………………………………………………………………………………………………….88

List of Tables

Table 1. Soil nutrient and cropping management data for four fields used The first column indicated which field was sampled one or two and the letter indicates if the cropping system was continuous alfalfa (A) or potato seed corn (B). The second column indicated the zone number that was sampled……………………………………………………………………………………………………….43

Table 2. Activity of four soil enzymes: Phosphatase, Tyrosine aninopeptidase, N-acetyl glucosaminidase and β-1,4-glucosidase (nmol/h/g) associated with two potato management systems in Mecosta County, MI in March 2009………………………………………………………………………..45

Table 3. Activity of four soil enzymes: Phosphatase, Tyrosine aninopeptidase, N-acetyl glucosaminidase and β-1,4-glucosidase (nmol/h/g) associated with two soil types in Mecosta County, MI in March 2009……………………………………………………………………………………………………….46

Table 4. Total and mean daily carbon mineralization (μg C/g soil) associated with two management systems and two soil types in Mecosta County MI, in March 2009…………………….47

Table 5. Absolute nematode population density (nematodes/100 cm3 soil) associated with two management systems and two soil types in Mecosta County MI, in March 2009………………48

Table 6. Absolute nematode population density (nematodes/100 cm3 soil) of two management systems and five trophic groups……………………………………………………………………….49

Table 7. Absolute nematode population density (nematodes/100 cm3 soil) associated with two soil types and five tropic groups in Mecosta County MI in March 2009……………………………50

Table 8. Soil characteristics of four fields sampled to determine the vertical distribution of Pratylenchus penetrans, Paratrichodorus pachydermus and Verticillium dahliae under Michigan potato systems…………………………………………………………………………………………………………………………51

Table 9. Absolute and relative population densities associated with Pratylenchus penetrans at multiple soil depths in four Michigan fields……………………………………………………………………………..52

Table 10. Absolute and relative population densities of the vertical distribution of Paratrichodorus pachydermus at three soil depths associated in four potato fields in Michigan………………………………………………………………………………………………………………………………….53

Table 11. Verticillium dahliae propagules per gram of soil associated with two soil depths in two potato fields in MI…………………………………………………………………………………………………………….54

Table 12. Vertical distribution of Paratrichodorus. pachydermus in two fields infected with CRSD in Michigan……………………………………………………………………………………………………………………55

Table 13. 16 Chemical treatments for Corky Ringspot Disease of potato control research in White pigeon MI………………………………………………………………………………………………………………………………………………………….56

Table 14. 7 Chemical treatments for Corky Ringspot Disease of potato control research in White pigeon MI………………………………………………………………………………………………………………………………………………………….57

vii

List of Figures

Figure 1. Four sites, Fields 1A, 1B, 2A, 2B and their associated management zones used for long-term alfalfa vs. potato-seed corn evaluation of soil enzymes, carbon mineralization and nematode community structure in relation to soil quality. Management zones are numbered arbitrarily for organizational purposes……………………………………………………………………………………..56

Figure 2. Potato tuber yields associated with 16 soil fumigant and non-fumigant nematicide treatments at White Pigeon MI in 2008……………………………………………………………………………………57

Figure 3. CRSD potato tuber symptoms associated with soil fumigant and non-fumigant nematicides treatments in White Pigeon MI in 2008……………………………………………………………….59

Figure 4. 2009 Grade A potato tuber yields associated with nematode control for seven treatments……………………………………………………………………………………………………………………………….61

Figure 5. 2009 TRV symptom expressions associated with seven chemical treatments……………………………………………………………………………………………………………………………….62

Figure 6. A-D. Possible tobacco rattle virus crystal structures seen in symptomatic tubers photos taken under Transmission Electron Microscopy…..……………………………………………………...63

viii

Chapter I.

Impact of Alfalfa on Soil-Borne Enzymes in Potato Systems

Abstract

Potatoes are commonly grown in two-year rotations with corn, wheat or other annual crops in Michigan. Due to the expense and lack of marketability, long-term cover crops like alfalfa are not usually included in the rotation. Four fields were selected in Mecosta County, MI to evaluate the impact of long-term alfalfa on soil enzymes (β-1,4-glucosidase, N-acetyl glucosaminidase, phosphatase, tyrosine aminopeptidase, and phenol oxidase), carbon mineralization potential and nematode community structure; compared to potato-seed corn rotation. The sites have been used for potato production since the 1970’s. Two of these fields, however, were taken out of potato production and used to grow alfalfa hay for ten years. These fields were returned to potato production in 2009 and 2010. The sites were sampled in 2009, for soil enzyme, carbon mineralization, and nematode community structure analyses. The research tested three hypotheses: 1) long-term alfalfa production results in greater soil enzyme activity in Mecosta and Covert sands compared to a seed corn-potato rotation, 2) continuous long-term alfalfa production results in greater in carbon mineralization than that associated with a potato-seed corn rotation, and 3) nematode community structure is more diverse in long-term alfalfa production compared to a potato seed corn rotation. The results indicated that phosphatase and tyrosine aminopeptidase activity was significantly greater in soil from the long-term alfalfa than soil from the potato-seed corn rotation. Carbon mineralization of soil from the continuous alfalfa was significantly greater (P < 0.05) than that from the potato-seed

1 corn rotation. There were significantly more omnivorous and herbivorous nematodes associated with the continuous alfalfa, compared to the potato seed corn rotations. These results indicate that soil quality can be influenced through use of a long-term cover crop such as alfalfa. They do not, however, indicate how long these benefits might last once a site is returned to a two-year potato rotation system.

Introduction

Over seventeen thousand hectares of potatoes are grown annually in Michigan, USA.

More than 30% are used for the production of potato chips. It is general practice for potato growers in Michigan to use a two-year rotation with corn or wheat. This rotation is done to elevate nutrients, and reduce pest issues that commonly arise in back to back crop rotations.

However, Taylor (2005) found that potato systems did not fully break down their plant residue that holds the microsclerotia of Verticillium dahlia for two years, and that even having a rotation of three years showed significant economic benefit by reduced early-die issues. Potato early die is the plugging of the vascular system of the plant by the microsclerotia Verticillium dahliae, which enters the plant through holes that the nematode Pratylenchus penetrans creates when it enters the root tissue. An increase in organic matter in the soil increased the rate of decomposition of the plant residue and microsclerotia (Taylor, 2005). The tillage alone associated with potato systems removes soil organic matter as well as increasing compaction.

Growers have become interested in soil quality and the potential role of long-term cover crops.

Soil quality can be defined as the ability of soil to resist degradation, and respond to management (Jansa, 2003).

Enzymes are essential catalytic chemicals in soil systems. They help degrade plant material into forms digestible by soil microbes, as well as facilitate nitrogen, phosphorus, and carbon cycling. Soil enzymes assays are used to determine the potential for soil to degrade or transform substrates (Sarapatka, 2003). Enzyme activity is important in the monitoring of soil quality because it is closely related to several soil quality parameters. Soil enzyme activity tends to change much sooner than other soil properties and is thus a good indicator of changes in soil quality. Soil enzymes have the ability to effect past management on soil biology. They also can be measured somewhat easier than some other soil properties (Sarapatka, 2003).

These enzymes are used, due to their relationship to microbial biomass and their sensitivity to management systems (Sarapatka, 2003). Five enzymes are of particular interest in soil systems:

β-1,4-glucosidase, N-acetyl glucosaminidase, phosphatase, tyrosine aminopeptidase, and phenol oxidase.

β-1,4-glucosidase (BG) is produced by hyphae of fungi such as A. niger (Lammirato,

2010), and is involved in the degradation of cellulose in soil by catalyzing the hydrolysis of celloboise to glucose. Cellulose is a sink for atmospheric CO2 and a source of organic carbon.

BG is believed to be a positive indicator of soil quality (Muruganandam, 2009).

N-acetyl glucosaminidase (NAG) is involved in the degradation of chitin, an abundant biopolymer found in most soils. Chitin is a source of organic N (Stevenson, 1994). NAG has been found in high amounts in the upper soil horizons of no-till systems. There is a strong positive correlation between the presence of fungal hyphae and the amount of NAG present in

3 the soil (Muruganandam, 2009), and NAG is a positive indicator of soil quality (Muruganandam,

2009).

Phosphatase (PHOS) is one of several enzymes involved in phosphorus and carbon cycling (Fernandez-Calvina, 2010). This enzyme is produced by fungi, plant roots, and other microorganisms, and hydrolyses phosphorus esters following organic matter’s initial break down (Sarapatka, 2003). PHOS is used to evaluate the level of ecosystem disturbance due to its sensitivity to treatments and climate, but not to daily weather variations (Madejon, 2001;

Sarapatka, 2003).

Tyrosine aminopeptidase (TAP) is involved in nitrogen and phosphorus cycling (Koch,

2007), and degrades proteins (Haase, 2008). High levels of this enzyme indicate a high capacity

+ + for converting plant-unavailable NH4 into NH3 (Koch O., 2007). High levels of CO2 inhibit the production of the TAP enzyme (Haase, 2008).

Phenol oxidase (Phenox) is produced by soil-borne fungi (Gallo, 2004), and is important in the degradation of lignin. It acts on phenolic substrates, which are oxidized to phenoxy, and participate in further reactions. Phenox declines in the presence of N additions (Gallo, 2004).

Compared to potato, corn, and wheat; alfalfa is a very deep-rooted crop (Rasse, 1998).

As a long-term cover, hay, pasture crop, it requires no tillage. Alfalfa production promotes soil aggregate formation and sequesters carbon and nitrogen in soil systems due to rhizobacteria associated with the roots of legumes that sequesters nitrogen (Robertson, 2006).

Unfortunately, alfalfa is also a good host of several nematodes that parasitize potato (Goodell,

1981), including Pratylenchus penetrans (root-lesion nematode). This species and Verticillium

4 dahliae are major components to the potato early-die complex (Rowe, 1985). The objective of this research was to compare enzyme activity nematode population and carbon mineralization across two management systems for a single snap shot in time to help evaluate the impact of long-term alfalfa on soil quality. Four adjacent fields were selected for the study. Two of the sites were historically in potato rotations, but planted to alfalfa for the last ten years, and two in a potato-seed corn rotation. We hypothesize that long-term alfalfa will result in different soil enzyme activity, carbon mineralization, and nematode population densities when compared to potato-seed corn rotation.

Methods

Site Description/Sampling: Four adjacent fields at latitude 43.565296, longitude -85.248395 were selected for the research (Figure 1). Fields 1A and 2A were planted to continuous alfalfa for ten years between 1999 and 2009. Field 1B and 2B were managed as a potato-seed corn rotation during the same period. Soil samples were taken in March 2009; sampling locations were determined using GIS and GPS software to equally sample the major soil textures of all four fields. Sampling zones were chosen by their size and representation of the field as a whole.

Soil nutrients (P, K, Mg, Ca, and Zn), cation exchange capacity, pH and organic matter were determined for the four fields (Table 1). Field 1A had two distinct soil types. Soil types indicated the percent of sand silt and clay that are present in each of the horizons these are determined by the USDA soil survey and are readily available online and on township soil maps.

The majority of the samples in 1A were taken in Covert sand with a 0-3% slope due to its prominence across the field. The parent material for Covert sand is sandy till which was

5 moderately well drained and with a water holding capacity of 10.7 cm. The typical soil profile includes 0-10 cm sand, 10-25 cm sand, 25-89 cm sand, 89-137 cm sand, and 137-152 cm sand.

(United States Department of Agriculture, 2010) The other soil type in this field was Mecosta

Sand with a 0-4% slope. The parent material for this soil was sandy and gravelly outwash. The drainage class was somewhat excessively well drained, with a water holding capacity of 8.1 cm.

The typical soil horizons were sand from 0-25 cm, gravelly sand from 25-50 cm, 50-56 cm is coarse gravelly sand, and 56-152 cm was extremely gravelly coarse sand. (United States

Department of Agriculture, 2010)

In field 1B, all samples were taken in Mecosta Sand with a 0-4% slope. The parent material for this soil was sandy and gravelly outwash. The drainage class was excessively drained, with a water holding capacity of 12 cm (United States Department of Agriculture,

2010). Field 2A, had two soil types Coloma Sand had a 0-6% slope, and made up two-thirds of the area. Coloma Sand had a sandy outwash parent material. It had excessively drained water holding capacity of 12 cm. The typical soil profile was 0-25 cm sand, 25-91 cm sand, and 91-152 cm sand. (United States Department of Agriculture, 2010) The second soil type was Mecosta

Sand with a 0-4% slope. Field 2B had one major soil profile and one minor soil profile. The major soil profile was Covert sand with a 0-3% slope. The minor component of this field was

Mecosta sand with a 0-4% slope. (United States Department of Agriculture, 2010)

Samples for enzyme activity, nutrient analysis, carbon mineralization and nematode community structure were taken at random in each of the major management zones in April

2009 at a depth of 20 cm, using a standard 2.54 cm in diameter soil core. A minimum of nine

6 sample sites were taken in each of the management zones sampled in all four fields. These management zones were determined by soil organic matter topography and size. Each zone was labeled with an arbitrary number for identification proposes. Crop roots from the previous year were also collected at sites corresponding to the other sampling sites at the same time.

These samples were taken at a depth of 20 cm using a trenching shovel and hand picking average sized roots during sampling to help determine the nematode population density

Nematode Population Density: Nematode population density was determined in each field using two methods. A 100 cm3 soil sample was processed for each of the zones sampled for nematodes using the centrifugal flotation method (Jenkins, 1964 ). Roots were processed using a shaker extraction method (Bird , 1971), briefly rinsed root tissue (1.0 g) that were collected for each sample, cut into 1-2 cm segments and was placed in a 125 ml flask with 100 ml of water a mixture of 10 ppm etxytehyl mercuric chloride and 50 ppm dihydrostreotomycin sulfate then it was incubated on a shaker table for 48 hours at 100 rpm. The liquid was then poured through a 400 mesh sieve, and the nematodes were washed off the sieve into a test tube. Both sets of samples were identified and counted using a Nikon TMS inverted microscope.

Nematodes were identified to the following trophic groups: bacterivores, carnivores, fungivores, omnivores, and herbivores. Population densities for each group were counted and recorded. Due to its importance in potato production systems, P. penetrans was recorded in both the herbivore group and as a stand-alone count.

Soil Enzymes: The Saiya-Cork (2002) protocol was used to measure enzymes activity. Samples from the four fields that were taken in April 2009, were used to make suspensions that were

7 prepared by homogenizing 1.0 g soil in 125 ml of 50 mM sodium acetate buffer pH 6, using a

Virtex 45 tissue homogenizer (Virtex Inc. Yonkers, NY). The suspensions were continuously stirred on a magnetic stir plate while 16 replicates of 200 μl aliquots were pipetted into 96-well microplates. Sixteen replicate wells were created for each of the five enzyme assays and zones sampled. All of the enzyme assays were fluorimetric, except the phenox, which was colorimetric. The flourimetric assays were conducted in black 96-well microplates and the colorimetric assays in clear microplates.

In the flourimetric assays, 50 μl of 200 mM substrate solution was added to each sample well. Eight blank wells per sample were created with 50 μl of buffer and 200 μl of sample suspension. Then eight quench control wells per sample were created with 50 μl of fluorescent standard (10 mM 4-methylunbelliferone) and 200 μl of the sample suspension. Eight substrate control wells per plate were also created with 50 μl of substrate solution and 200 μl of buffer.

Finally eight reference standards per plate were created with 50 μl of fluorescent standard and

200 μl of buffer. The assay plates were incubated in the dark at 13°C for 24 h. The reaction was terminated by adding 10 μl of a 1.0 M NaOH solution to each well to raise the pH to 9.0, the optimal pH for the fluorescence of these substrates. Fluoresence was measured with 365 nm excitation and 460 nm emission filters using a microplate fluorometer (Fluoroskan II, Thermo

Labsystems, Waltham, MA). For the phenol oxidase analysis, each sample well received 200 μl of sample suspension and 50 μl of 25 mM L-3,4-dihydroxypjenylalanine (L-DOPA) as the substrate. The negative control wells contained 200 μl of acetate buffer and 50 μl of L-DOPA.

The blank wells contained 200 μl of sample suspension and 50 μl of acetate buffer. The colorimetric assay plates were incubated for 24 h at 13°C in a limited light environment.

Enzyme activity was determined spectrophotometrically by measuring absorbance at 460 nm

(Spectranmax Microplate Spectrophotometer, Molecular Devices Inc., Sunnyvale, CA). Enzyme activities were corrected for moisture and were reported in nmol/h/g (Grandy, 2009).

Soil enzyme activity was compared for the cropping systems and soil types. There were eight replicated samples for the potato-seed corn system and seven for alfalfa. An eighth data set was generated for alfalfa using the missing value equation (Cochran, 1937). Soil enzymes were also compared across soil types. Two main soil types were sampled in the four fields.

Seven samples were taken in Mecosta sand and six samples were taken in Covert sand. One missing value was generated for the Covert sand by using the missing value equation.

Carbon mineralization: Carbon mineralization was determined by measuring soil respiration over time (Grandy, 2007 ). Fifteen grams of soil from each site that was sampled in all four fields was placed in glass 50 ml vials. Samples were brought up to 60% water holding capacity by weight. The vials were fitted with septa and placed in an incubator at 25°C. Sampling of each vial’s CO2 was performed three times a week for three months. Prior to sampling, the CO2 analyzer was calibrated using an empty vial and septum that was flushed with 10% CO2 for one minute. Using a syringe, three readings were then taken for each of the following volumes, 0.1 ml, 0.2 ml, 0.3 ml, 0.4 ml, and 0.5 ml. These samples were injected into an infrared gas absorb ion analyzer (IRGA) that recorded the amount of CO2 in ppm. The vials with soil had their septum removed and were allowed to equilibrate with atmospheric CO2 levels for 30 minutes.

The septum was then replaced. At time zero the CO2 value was obtained by extracting 0.5 ml.

From each bottle and injecting the gas removed into the IRGA. This was repeated again at 30

9 minutes and 60 minutes. The samples were returned to the incubator until the next sampling date. Following sampling, the data were analyzed to determine the respiration potential over the time interval (Robertson, 1999). The results were compared across two variables cropping system and soil type.

Data Analysis: Data were analyzed using a two-factor student T test, comparing both soil type and management system. When comparing soil enzymes in the same rotation, but in different soil types, there were differences in the number of data points. Missing data points were

adjusted using the missing data formula , where a is the number of

treatments, b is the number of blocks, T is the sum of items with same treatment as missing item, B: is the sum of items in same block as missing item, and S is the sum of all observed items. (Cochran, 1937) A 95% confidence interval was assumed despite the variability of soil systems.

Results

Soil Enzyme Activity/Management System: When the activities of the four enzymes were compared across management systems, phosphatase (PHOS) and tyrosine aminopeptidase

(TAP) showed significantly greater activity in the long-term alfalfa compared to the potato-seed corn rotation (P=0.003) and, (P=0.024) respectively. The N-acetyl glucosaminidase (NAG) and β-

1,4-glucosidase (BG) were not significantly different in the two management systems system

(Table 2).

Soil Enzyme Activity/Soil Type: The enzymes were then compared between the two major soil types that made up the majority of the sampling sites. Only NAG showed a significantly higher amount of enzyme activity (P=0.014). The other three enzymes PHOS, TAP, and BG were not significantly different (Table 3). This was not unexpected and these comparisons were done mainly to determine if this variable played a role in the amount of enzyme activity present in the fields.

Carbon Mineralization: Soil enzyme carbon mineralization was measured to determine the amount of carbon in the two management systems. Carbon mineralization was compared across both management systems as well as both soil types. This was done as total amount of carbon that was released as CO2 throughout the three month experiment, as well as on a daily basis (Table 4). There was significantly (P=0.029) more carbon released from the continuous alfalfa system than the potato-seed corn rotation, and there was no significant (P=0.866) difference in total carbon released across soil types.

Nematode population density: Nematode population was compared three different ways. The total nematode population was determined across management system and soil type. The nematodes were also grouped into five different trophic groups based on their feeding habitats and compared across management system and soil type. Finally the Pratylenchus penetrans were removed from the herbivorous nematodes group and compared across management system and soil type. Total nematode population was significantly (P=0.017) affected by cropping system, but not (P=0.364) by soil type (Table 5). When the nematodes were split into trophic groups and compared across management systems the omnivores, herbivores, and P.

11 penetrans showed significantly, (P=0.000), (P=0.000), and (0.005) respectively, greater populations in the alfalfa system than in the potato-seed corn rotation. When nematode population densities were compared between the two soil types, no statistical differences were observed in any of the five trophic groups or P. penetrans (Table 7).

Discussion

Managment system effects on soil enzymes: The results of this study indicated that continuous alfalfa increased soil enzyme activity of phosphatase (PHOS) and tyrosine aminopeptidase (TAP) when compared to an intensely managed potato-seed corn system.

PHOS is involved in phosphorus and carbon cycling (Fernandez-Calvina, 2010) and is often used as an ecological disruption indicator of soil (Madejon, 2001). One reason for lower amounts of this enzyme in the potato-seed corn rotation could be that it has been shown that there is a strong correlation with carbon concentration from organic amendments and the stimulation of soil enzyme activity. Inorganic fertilizers have also been shown to suppress soil enzyme synthesis (Sarapatka, 2003). PHOS production can be depressed by the addition of phosphorus fertilizers (Sarapatka, 2003). Phosphorus fertilizers are applied several times a year in corn and potato production but are rarely applied to an alfalfa management system.

TAP enzyme activity had a significant increase in the continuous alfalfa system. This may be due in part to several factors, including the microbial health of the soil, soil organic matter present, and the quality of soil structure. TAP is involved in the nitrogen cycle. Sufficient

TAP in a soil system enhances the transformation of unavailable nitrogen into NH3 (Koch, 2007).

TAP is produced by soil microbes such as soil borne fungi. These organisms require several

12 conditions to be successful, including adequate energy source and proper environment for growth and reproduction. One food source of many microorganisms is soil organic matter

(SOM). The alfalfa management system had significantly higher amounts of carbon mineralization than the potato seed-corn rotation. Carbon mineralization can be directly tied to the amount of active carbon that is in the soil system. Another factor that affects the health of the soil microbial community is soil structure, which is impacted tillage, fertilizer applications, and the annual freeze-thaw cycle that occurs in Michigan. Haynes (1996) and

Grandy (2007) showed that less tillage reduces the carbon-holding capacity of soil. The reduction of tillage and the increase in leguminous crops increases soil aggregation, which can increase the amount of active and resistant carbon held in the soil (Grandy, 2007). This is due in part to the larger amount of organic matter that is deposited into the soil profile through large rooting systems as well as polysaccharides that are known to stabilize and promote soil aggregation. Aggressive tillage promotes the release of carbon from the soil into the atmosphere in the form of CO2 by increasing the amount of O2 in the system a limiting factor for many soil borne microbes. Tillage also decreases the diversity of microbial life in the soil by degrading the structure of the soil system, thus limiting the habitats available. The decrease in structure through tillage also affects water holding capacity and porosity of the soil, both of which are promoters of soil microbial health. Fungal hyphae, which produce soil enzymes, are also broken up by tillage. These fungal hyphea and soil borne microbes release enzymes into the soil, and helps form an environment that is conducive to the increase of soil quality. (Jansa,

2003)

One important contributor to soil quality is the amount and type of soil organic matter present. Soil organic matter can come from 1) residue left of the soil after crop harvest, 2) organic matter additions by the grower, 3) from historic organic matter additions as is the case with soils such as muck. Over the ten-years that the continuous alfalfa fields were out of potato production, they received one application of non-composted beef manure from feedlot, per year. Manure contributes of phosphorus, carbon and nitrogen to the system, and is preferred by soil microbes as a food source over synthetic forms of these elements (Gallo,

2004). The two fields that remained in potato production only received synthetic forms of phosphorus and nitrogen. Manure tends to be more structurally complete that synthetic fertilizers. This is known to influence the amount of soil organic matter (SOM) that can build up in the soil (Robertson, 2006). Increases in SOM provide nutrients for enzyme forming soil microbes that are limiting factors for growth in systems that only receive synthetic fertilizers.

These synthetic fertilizers are not as complex as manure or residues from leguminous crops such as alfalfa and can even reduce the formation of certain soil enzymes such as phosphatase

(Sarapatka, 2003). Another factor that influenced the quality of soil organic matter in the soil was the amount of residue that the alfalfa plants left in the soil with their extensive root systems as well as the above ground portion of the plant.

The last factor that might have affected the amount of TAP was the ability of the long- term alfalfa rotation to resist degradation by the freeze thaw cycle common in Michigan. In the winter months, soils that are protected by a layer of organic matter have a much weaker freeze thaw effects on the structure of the soil. Soil moisture in micro and macro pores in the soil during the winter goes through a freeze thaw cycle that will break apart soil aggregation and

14 lead to an increase in the loss of carbon and thus a reduction of habitat and food source for soil microbes. (Robertson, 2006)

There was no significant difference between the two management systems in NAG levels. NAG is involved in the degradation of chitin. This indicates that the amount of chitin in both systems will not be degraded at a higher rate in either of them. There was no significant difference between the two cropping systems in β-1,4-glucosidase. BG is involved in the breakdown of cellulose, a large sink for atmospheric CO2. NAG and BG activity levels are positively correlated with an increase in SOM (Shi, 2011). The current study does not contradict this conclusion, but due to lack of statistical significance it does not support it either. The lack of statistical difference could be due, in part, to timing of sample collection, and soil temperature. The fields were sampled during April in Michigan when microbial activity may be suppressed due to soil temperatures. Shi (2011) found a difference in microbial levels when sampling later in the spring, when many soil microbes are more active due to warmer soil temperatures. Soil microbes decompose crop residue and product of compounds such as soil enzymes. Soil temperature might also be affected by the direct exposure of the ground to radiant sunlight the potato-seed corn soils which were not protected by a layer of organic matter, warmed up slightly more rapidly. The alfalfa field’s vegetation might have affected the solarization of the soil causing less microbial activity to occur than it might have at the same temperature as the potato-seed corn rotation.

Soil Type Effects on Soil Enzymes: When the soil enzyme levels were compared across soil type, NAG showed significantly greater enzyme activity in the Covert Sand (P=0.014), while

TAP, PHOS, and BG all were statistically the same across both soil types. The soil types were similar, and lack of difference in enzymes was expected. The only physical difference between two soil types was that Mecosta sand was better drained and had a slightly coarser texture compared to the smaller particle size than the Covert sand. This slight difference might have allowed more attachment sites for microbes as well as allowing a larger water holding capacity that would promote the health and growth of enzyme creating bacteria and fungi. While we did not see any significant differences between BG, PHOS and TAP enzyme levels in the two soil types the Covert Sand was slightly higher in activity across all three enzymes that showed no difference.

Soil Carbon Mineralization: When carbon mineralization was compared by soil type and cropping systems, there was no significant difference between the two soil types, but cropping system did make a significant difference in the amount of carbon mineralized and release of

CO2. Both soil types were similar, with the same amount of clay, silt, and loam, Therefore have a similar water holding capacity. Two of the key factors impacting carbon mineralization are physical and chemical structure of the soil. The physical factor that contributes most to mineralization is aggregation (Grandy, 2007 ). Aggregates are created, and held together by, microbial secretions such as polysacarides and enzymes (Grandy, 2007 ). When aggregates are tightly packed together in undisturbed systems, oxygen is very slow to diffuse into the aggregates. Creating an anaerobic, O2 limited system (Robertson, 2006). As aggregates break down mechanically, O2 flushes the system. The microbes that are normally suppressed by lack of O2 are stimulated, and there is a burst of carbon mineralization in the form of CO2. The

16 chemical factors that contribute to mineralization are the amount of carbon in the active, passive, and resistant pools. The active carbon pool is the carbon that is the shortest lived; it is the first to be consumed by microbes and is readily available in most systems (Grandy, 2007).

The passive carbon pool is longer lived than the active pool, and is made up of longer carbon chains that are harder for soil microbes to metabolize. The resistant carbon pool is the longest lived in the soil, and is made up of substances like humus that are extremely long carbon chains and need enzymes to cleave them in order for soil microbes to utilize them as a food source

(Grandy, 2007).

At least one enzyme involved in the carbon cycle was present in greater amounts in the continuous alfalfa rotation. The alfalfa rotation had no mechanical disruption over a ten-year period degrade soil aggregates. In contrast the potato-seed corn rotation was tilled aggressively several times a year, breaking up aggregates and flushing the system with O2. This tillage stimulated the release of soil carbon from the active and passive soil carbon pools, decreasing the amount of CO2 present for release in a laboratory setting.

Nematode Community: Nematodes are aquatic organisms that live in the thin water films surrounding soil particles and in host tissue. Some groups have a wide host range, feeding on bacteria, fungi, algae, or plants, while others are carnivorous or omnivorous. Historically, nematodes were widely recognized 1) as pathogens of infectious disease of plants, animals and humans, and 2) vectors of plant viruses. (Brown, 1995) More recently, the trophic groups associated with different ecological systems used to gauge the health of a crop system.

Nematodes role in nutrient cycling is also better understood. Neher (2010) showed that

17 decomposition rates were 1.4-1.9 times faster in conventional tillage systems than in no-till systems, where residues remain on the soil surface and are not incorporated (Neher, 2010).

Nematodes play a role in the decomposition of organic matter, and the increase amount of organic matter that is in the soil will promote habitats that are favorable for the nematodes and the food sources of the nematodes.

Nematodes play a role in nitrogen cycling by excreting ammonium as a byproduct after consuming prey, mainly bacteria, that have a lower C:N ration than they need. Bacterivourous and predatory nematodes contribute between 8% to 19% of nitrogen mineralization in conventional and integrated farming systems, respectively (Neher, 2010). In all trophic groups, population densities were higher in the continuous alfalfa, than in the potato seed corn rotation. There were statistically higher populations of omnivores and herbivores in the continuous alfalfa rotation compared to the potato-seed corn rotation. Very few fungivores or carnivores where associated with either of the two management systems. Overall, there was a higher nematode population density in the continuous alfalfa than associated with the potato- seed corn rotation. One reason for this is that perennial plants have root growth that is more ephemeral than annual crops, and more closely resemble a natural systems than cropping systems (Neher, 2010). Also, the above ground plant material may have had a protective effect and increased the survival over-wintering individuals. The populations in the potato-seed corn might have been diluted by spring tillage that was performed prior to sampling. As well as fumigation applications that are common on a biannual bases determined by the population densities of P. penetrans and Verticillium dahliae.

There were significantly higher numbers of P. penetrans in the continuous alfalfa rotation, but no significant difference between soil type. This we believe can be attributed to the lack of disruption of the alfalfa system over a ten-year period. Although P. penetrance is known to thrives in disturbed systems, the fields that were in a potato-seed corn rotation were biannually fumigated with metam sodium, lowering nematode populations.

Conclusion

It was anticipated that there would be significant differences in soil enzyme, carbon mineralization, and nematode community structure between the continuous alfalfa rotation, and the potato-seed corn rotation. These differences are believed to be due to tillage system, input, and crop residue differences. A statistically significant increase in PHOS and TAP activity in the continuous alfalfa was seen while there was no significant increase in NAG or BG. In the enzymes that were not significant the continuous alfalfa did show a trend towards having higher enzyme activity. When enzyme activity was looked at across soil type we saw that NAG was the only enzyme that showed a significant increase in the Covert sand. This lack of difference in the majority of the soil enzymes activities was attributed to the similarity of the two soil types sampled.

It was hypothesized that carbon would have significantly higher mineralization rates in the continuous alfalfa rotation, due to the ability of the soil to build up a greater active and passive carbon pool in soil aggregation in the absence of tillage. There was a significant difference between the two management systems, with higher carbon mineralization taking place in the continuous alfalfa rotation. There was no significant difference in carbon

19 mineralization between the two soil types. This could be attributed to the similarity of the soil texture. Finally, we hypothesized that there would be statistical difference between the alfalfa rotation and the potato-seed corn rotation in the nematode community structure. The nematode trophic groups, omnivores and herbivores, had statistically higher population densities in the continuous alfalfa system than in the potato-seed corn rotations. The bacterivores, fungivores and carnivores were not significantly different from one management system to another, but the continuous alfalfa had higher populations of all three of these trophic groups. There was a significantly higher P. penetrans population in the alfalfa rotation than in the potato-seed corn rotation. This we believe can be attributed to the biannual occurrence of fumigation in response to potato early-die, as well as dilution of populations by spring tillage in the potato-seed corn rotation. The research indicates that long-term alfalfa in a potato cropping system has the ability to replace carbon in soil as well as increase the activity of some of the soil enzymes that are important in the carbon, phosphorus, and nitrogen cycles. It also showed an increase in the populations of P. penetrans and omnivorous nematodes.

Chapter II

Vertical Distribution of Paratrichodorus pachydermus, Pratylenchus penetrans (Nematoda), and Verticillium dahliae (Mycota) in Michigan Potato Soil Systems

Abstract

Potato production is a multi-billion dollar a year commodity in the U.S. and is in the top ten of food crops grown in MI. The goal of growers is to produce high yields of high quality potatoes that will return the highest amount of profits possible. Several diseases and environmental challenges interfere with this goal. Two of these challenges for Michigan potato growers are Potato Early-Die (PED) and Corky Ring-Spot Disease (CRSD). PED is caused by an interaction between the root-lesion nematode (Pratylenchus penetrans Cobb, 1917), and

Verticillium dahliae Kleb (Mycota). This infectious disease and associated symptoms reduce tuber yields up to 46%. The stubby-root nematode (Paratrichodorus pachydermus Seinhorst,

1954), on the other hand, is generally not a factor in yield reduction. It is, however, a contributing factor in decreased tuber quality. Paratrichodorus. pachydermus is a vector of

Tobacco Rattle Virus (TRV), which causes CRSD. This disease was detected at two sites in MI.

Research was conducted in the fall of 2010 to determine the optimum sampling depth for detection of (P. penetrans, P. pachydermus, and V. dahliae) associated with these two diseases.

Four Michigan potato fields were sampled at two soil depths (0-15 cm, 15-30 cm) or three soil depths (0-15 cm, 15-30 cm, and 30-45 cm). The research indicated that in the fall P. pachydermus is predominantly located below a 15 cm soil depth. Population density of P. penetrans was found to be significantly higher in the upper 15 cm in a field that was sampled that had potatoes growing the year it was sampled; whereas, significantly higher population

21 densities were found in the 15-30 cm depth in fields that had seed corn the year it was sampled. We also found that there were no significant differences between the population densities of V. dahliae at either of the soil depths sampled.

Introduction

The United States potato crop has an estimated value of $3.45 billion, and potatoes are grown on 445,000 hectares nationally. Michigan grows about 17,400 hectares of potatoes annually (NASS, 2011). The majority of these acres are sampled by growers and agronomists for plant parasitic nematodes and Verticillium dahliae Kleb (Mycota). These samples are taken from a range covering a soil depth of 0-20 cm, a depth thought to give the best estimate of risk of Potato Early-Die (PED) disease complex. PED is caused by the interaction of the root-lesion nematode (Pratylenchus penetrans Cobb) and Verticillium dahliae.

Potato early-die is common throughout U.S. potato production, causing yield losses up to 46% (Rangahau, 2003; Johnson, 2000). Pratylenchus penetrans invades the basal roots of young potato plants immediately upon emergence (Chen, 1995). This invasion is followed by colonization of underground and above ground tissue. Pratylenchus penetrans is a migratory endoparasite that moves out of the soil profile and into root tissue. Its migration through root tissue results in downward movement of the nematode as roots grow. When the roots degrade, these nematodes are left at soil depths out of the reach of conventional sampling and soil fumigation practices. Pratylenchus penetrans, like P. pachydermus, has a wide host range, and can cause root stunting and yield loss (Baldridge, 1998).

Verticillium dahliae infects plant tissue through the roots. It moves into the vascular system where the mycelium grows and reduces the flow of water and nutrients to above ground tissue. Verticillium dahliae often uses necrotic lesions caused by P. penetrans as infection points of entry into root tissue. The blockage of the vascular system can lead to stunting and early death of the plant, severely limiting the potential tuber yield.

The vertical distribution of plant parasitic nematodes Paratrichodorus pachydermus and

Pratylenchus penetrans has been studied extensively. This lead to the standardization of a 20 cm soil sampling depth for determining nematode population densities. Pudasaini (2006) showed that in four different field crops, P. penetrans had the highest populations at a 10-30 cm soil depth. MacGuidwin (1991) found that P. scribneri was not distributed uniformly in the top 38 cm of the soil profile. Few nematodes were found near the soil surface and few were found below 30 cm. Both of these species showed similar vertical distribution to that found for

Pratylenchus brachyurus, which had the highest population at 15-30 cm soil depth. High populations of P. brachyurus, however, were found in the 0-15 cm layer on some sampling dates (McSorley, 1990). When P. penetrans was evaluated under raspberry in the summer, the highest populations were in the 5-10 cm range (Forge, 1998). When soils were evaluated for vertical distribution under millet (Panicum ramosum L.), P. brachyurus was found to be in the highest population at the 45-75 cm depth. On soybeans [Glycine max (L.) Merr. Cv ‘Custer’] when there was 78-79% sand, 6% silt, and 15-16% clay (Brodie, 1976). These results were different from McSorley’s work that showed P. brachyurus in highest population at the 15-30 cm depth (1990). Pratylenchus nanus was shown to be at its highest population in the spring of the year at the 0-10 cm depth (Bell, 2001).

Trichodroid nematodes, by comparison, are found deeper than Pratylenchus spp, they between 10-40 cm (Boag, 1987). Paratrichodorus minor was at its highest population at a depth of 30-45 cm in Florida in summer under maize (McSorley, 1990). This species was found at their highest populations at a depth of 10-20 cm in the spring under pasture conditions in

New Zealand (Bell, 2001). Trichodorus christiei was found in its greatest population under soybeans following millet at a depth of 30 cm where the soil was 83% sand, 5% silt, and 12% clay. The highest populations occurred in December to March when soil temps were 11-17°C and soil moisture was 18-23% by volume (Brodie, 1976).

Two nematode species, P. penetrans and P. pachydermus, and one fungus, V. dahliae were evaluated in this research. Paratrichodorus pachydermus Seinhorst, 1954 (Stubby-root nematode) is an ectoparasite that tends to prefer cool wet soils (Bird, 1967). It is a concern to growers because of its broad host range and ability to vector plant viruses. It is a vector of the rod shaped tobravirus that causes Corky Ring-Spot Disease of Potato. This disease causes a loss in quality of the crop, but usually not a yield reduction (Wernette, unpublished). High population densities of P. pachydermus can also lead to yield loss by stunting root growth by feeding on the root tips.

Despite sampling and management options such as fumigation and crop rotation, tuber yield is lost annually to nematodes, viruses and fungi. This calls into question the reliability of the sampling procedure for nematodes. Several factors, including soil type, management history, and crop rotation can affect the population densities of these pests. One variable that can be studied is vertical distribution. It is essential to know where the highest proportions of

24 plant parasitic nematodes reside. Potato seed pieces are generally planted at 15-20 cm, depth corresponding with the low end of the current soil sampling depth procedure. Potato roots, however, can reach a soil depth of 60 cm (Durrant, 1973). Tillage in the upper 30 cm of soil tends to homogenize the soil and associated pathogens (Robertson, 1995). Tillage results in an equal distribution of nematodes and V. dahlia microsclerotia. Current sampling methods may not to provide an accurate estimate of the populations of pests that lie below 30 cm, with the potential to infect root tissue.

Soil texture plays a large role in nematode community structure and vertical distribution. Many plant parasitic nematodes prefer sandier soils to soils that are higher in clay

(Kable, 1968; Thomason, 1959). As soil type changes, so does water holding capacity and soil organic matter content. Soil compaction also plays a role in the speed at which migratory nematodes move through soil. Very coarse textured soils like sand have less water holding capacity, but larger pores that allow nematodes to move though the soil, more easily than in a soil type with a large amount of clay and relatively small pores. The amount of organic matter in a soil also affects the numbers and types of nematodes present. Most soil microbes feed on soil organic matter. Soil microbes like bacteria and fungi are a food source for fungal and bacterial feeding nematodes.

This current research project was done to: 1) look at the differences among three plant pest species at different depths in the soil profile and 2) determine if the soil sampling depth commonly used is representative of the actual nematode and fungus populations associated with potato production system. It was postulated that greater levels of P. penetrans and P.

25 pachydermus below the level of standard sampling and higher levels of V. dahlia in the upper layers of the soil due to the microsclerotia would be left on the soil when the above ground potato biomass degraded.

Methods

Site Selection, Location and Tillage: Soil sampling was conducted in the October and

November of 2010 in four MI potato fields. Two of the sites selected were known to be infested with corky ringspot disease of potato, and two were part of a 2010-2011 fumigation research project (Table 8). Field 1 is located in St. Joseph Co. (41.770256N, 85.683557W), Field 2 in

Saginaw Co. (43.371647N, 84.266406W), and Field 3 in Tuscola Co. (43.530707N, 83.22839W), and Field 4 in Mecosta Co. (43.514355N, 85.365533W).

In 2009, the field was tilled in the spring with a chisel plow at 31 cm and then planted to potatoes. After the potatoes were harvested, the ground was chisel plowed again. Corn was planted in the spring of 2010 and the red clover broadcast spread over the corn. Field 1 was in corn stubble inter-seeded with red clover in fall 2010. No tillage was performed prior to sampling in October of 2010.

Field 2 was planted to field corn in 2009. Following harvest, it was ripped (deep chisel plowed) at a depth of 38 cm, then fumigated with metam sodium at 374 liters/hectare. Prior to planting potatoes 2010, the seed bed was prepared by chisel plowing at a depth of 31 cm, then tilled using a disc at a depth of 20 cm. The potatoes were harvested in October and the field was ripped at 38 cm and tilled using a disc at a depth of 20 cm. Rye was planted as a cover crop.

Field 3 was planted to field corn in the spring of 2009. The following spring the corn stubble was chisel plowed at 31 cm and tilled with a disc at a depth of 20 cm prior to planting another crop of field corn. Following harvest, the corn stubble was tilled using a disc at a depth of 20 cm. At this point, nematode samples were taken.

Field 4 was disced at a depth of 20 cm in the spring of 2009. The field was moldboard plowed to a depth of 37 cm and potatoes were planted. Two in-season cultivations were performed, one at a depth of 8 cm between rows and a second at a depth of 20 cm. After potato harvest in the fall of 2009, the field was disced at a depth of 20 cm. Rye was broadcast spread across the field and the field was field cultivated at a depth of 15 cm. In the spring of

2010, the field was cultivated twice at a depth of 15 cm. Seed corn was planted and cultivated to a depth of 8 cm, at which time nitrogen was applied as a side dressing. In the fall of 2010, the corn was harvested and the stalks shredded. The field was disced once at 31 cm, and 2 weeks later it was disced again at 31 cm. Soil samples were taken just prior to soil fumigation in the fall of 2010.

Soil Sampling: Fields 1 and 2 were both sampled with a 2.54 cm diameter soil probe at three different depths, 0-15 cm, 15-30 cm, and 30-45 cm. Field 1 was split into five equal sections. One sample was taken per section with 20-30 random probes per sample using a transect across each section. Field 2 was split into ten equal sections. Field 2 was 15 ha while

Field 1 was 5.5 ha. The same procedure for sampling was performed in this field in each of the

10 sections.

Soil samples were taken in Fields 3 and 4 at two depths, 0-15 cm and 15-30 cm. Plots

7.6 m wide by 76.2 m long were established and sampled from, prior to fumigation. Twelve samples were collected by taking 20-30 probes per sample. A random zig-zag pattern across each plot was used at each depth. The 0-15 cm depth was taken using a standard Nematology cone shaped soil probe. Samples from the 15-30 cm depth were collected using a standard agronomic soil probe that is 2.54 cm in diameter. The 15-30 cm sample was taken from the same probe insertion point as the 0-15 cm sample. Samples from the two separate depths were placed in separate bags, labeled, and returned to Dr. Bird’s laboratory at Michigan State

University for processing.

Soil Borne Pathogen Analysis: Nematode population densities were determined using the centrifugal flotation method (Jenkins, 1964 ). A homogenized 100-cm3-soil soil sample was processed for each sample. Paratrichodorus pachydermus and P. penetrans were identified and counted using a Nikon TMS inverted microscope (40x). Pictures of the specimen were taken and placed on a CD-ROM drive and added to the Michigan State University Department of

Entomology collection. Verticillium dahliae propagules/ g of soil were determined for all samples from Field 3 and 4, using the wet sieve method by the Michigan State University

Diagnostics lab using the procedure described in by Nicot (1987). Fields 1 and 2 were not analyzed for V. dahliae because it was not originally part of the research.

Data Analysis: Data were analyzed using a two-factor student T test, comparing P. pachydermus, P. penetrans, and V. dahliae across the sampling depths in each individual field.

A 95% confidence interval was assumed, despite the variability of soil systems. For each site at each depth, a percentage of each nematode species was calculated by

Results

Pratylenchus penetrans: Root-lesion nematodes had higher population densities at soil depths >15 cm compared to the <15 cm depth, in three of the four locations (Table 9). In Field

1, 75% of the root-lesion nematodes recovered were from a soil depth of <30 cm. While in

Field 2, 89% of the root-lesion nematodes recovered was found at a soil depth of <30 cm. Most of the root-lesion nematodes were found below 15 cm (67% and 76% in Fields 3 and 4, respectively). Fields 3 and 4 were not sampled at soil depths below 30 cm. There were, however, significant differences between population densities of root-lesion nematodes between the 0-15 cm and 15-30 cm soil depths (P=0.006, P=0.001). This was not true for Field 1 or 2, where the difference in nematode numbers at the upper two sampling depths were not significantly different. The 30-45 cm depth had a significantly lower population density of root- lesion nematodes in Field 2, but there was no significant difference between densities at 0-15 cm depth and 30-45 cm depth in Field 1.

Paratrichodorus pachydermus: Stubby-root nematodes were found in three of the four fields sampled (Table 10). In these three fields, 100% of P. pachydermus were found below 15 cm. In Fields 1 and 2, 66% and 86% of the P. pachydermus were found below 30 cm, respectively. Of the P. pachydermus detected in Field 3, 100% were found below 30 cm.

Densities in nematode numbers at all three depths were significantly different from one another for Fields 1 and 2 (Table 10). Nematode population densities were too low to show significant differences in Field 3.

Verticillium dahliae wilt fungus: There were no significant differences (P=0.386,

P=0.777) between V. dahliae densities measured as propagules/ gram of soil at 0-15 cm and the 15-30 cm depth in Fields 3 or 4. The mean propagules/g of soil of V. dahliae ranged from 1.6 to 2.2 and 4.6 to 5.2 in Field 3 and 4, respectively at each depth (Table 2.4).

Discussion

Pratylenchus penetrans: In the fall, Pratylenchus penetrans is most common in the upper 30 cm of the soil. This is consistent with other research (Pudasaini, 2006; McSorley,

1990; MacGuidwin, 1991). There was some variability in the population density and its location in the soil profile from field to field. This may to be due to differences in management practices, cropping history, and temporal effects. MacGuidwin (1991) offered two explanations for the differences in nematode distribution 1) Nematodes will migrate vertically to seek more favorable conditions and 2) Resource abundance might differentially favor nematodes at various depths (Hesling 1967, Mojtahedi 1989). Fields 1 and 2 had the greatest proportion of the P. penetrans poulations in the upper 30 cm of the soil. This was expected as it is believed that the majority of nematodes congrag ate where most resources are located. For a plant parasitic nematode, this is in the rhizosphere of the host species. Field 2 was planted to potatoes prior to sampling and Field 1 was planted to corn. The corn would have created a large mass of roots in the 10-30 cm area through-out the growing season, when the nematodes

30 were the most active, and at their highest population densities. There was however no significant diffrence between densities of 0-15 cm and 15-30 cm depths in either of these fields. When we looked at Fields 3 and 4, they both had significantly greater P. penetrans population densities at 15-30 cm depth than at 0-15 cm depth. The biggest difference between

Field 2 and Fields 3 and 4 was the crop that was grown in 2010, pror to sampling. Rawsthorne,

(1986) indicated that vertical distribution was related to the pattern of the host roots. Field 2 had a crop of potatoes (cv FL 1879) in 2010, while Fields 3 and 4 were both planted to corn.

The rooting patterns of these crops varies, with potatoes having a larger proportion of their roots closer to the soil surface <30 cm deep (Lesczynski, 1976), while corn is a slightly deeper rooted crop (30-40 cm) (Hilfiker, 1988). One major item to consider is that potato growers most often sample their fields for fumigation managment decisions because they are planning to plant potatoes. Sampling is rarely done following potatoes but postponed until the fall or spring before potatoes are planted. The results we arrived at in Fields 3 and 4, are closer related to the conditions a field would be at the time of sampling thus making them a better model to base sampling depth decisions on, than the results from Field 2. Field 1 was entirely different than the other three fields, in that it was not tilled prior to sampling. This would have eliminated the stirring of the soil and the vertical mixing of soil and thus the dilution of the nematodes (Robertson, 1995). Tillage during the season also has a chopping effect on the roots that might slow or stop the movement of P. penetrans into root tissue by eliminating the excretion of root exudates that the nematodes use for root location.

Paratrichodourus pachydermus: Trichodorid nematode species are found deeper than many other nematodes in the the soil profile (Brodie, 1976). These nematodes can be very

31 important from a economic and product quality standpoint. They vector rod and virus particles that can cause yield loss and quality issues (Taylor, 1970; Hoy, 1984). P. pachydermus is a known vector of tobacco rattle virus, the cause of corky ring-spot of potato (Taylor, 1970).

Corky ring-spot has been reported and studied in both Fields 1 and 2, but very few virus vectors were observed (Wernette unpublished; Kirk, 2008). When this nematode was looked at in these two fields, two things were evident: 1) 100% of the P. pachydermus found, were below the 15 cm depth; 2) Field 1 had 66% of its P. pachydermus population below 30 cm, while field

2 had 86% of the P. pachydermus population residing below 30 cm. This is well outside the standard sampling depth protocal. P. pachydermus was also present in low numbers (<1/100 cm3 soil) in Field 3; All P. pachydermus detected, however, were below 15 cm. This field was not sampled below 30 cm so the answer to the question, if this field follows the same trend of having the majority of the P. pachydermus below 30 cm can not be determined. It is well known that P. pachydermus is a migratory nematode that prefers cool wet soils. As soils dry out and warm up through the growing season, nematodes move deeper into the soil. The following spring they move back towards the soil surface (Bird 1967). Our results are consistant with other work that says that trichodorids are generally located between 10-40 cm deep and that population densities change due to temporal factors, usually associated with soil temperature and moisture content (Boag 1987, Bird 1967). P. pachydermus has a very wide host range, including all of the common crops grown in MI as well as most common weed species (Bell 2001).

Verticillium dahliae: No differences were seen in V. dahliae levels between the two depths in either Field 3 or Field 4. Tillage of the field prior to sampling could very well have

32 homoginized the free microsclerotia within the soil profile. Any microsclerotia that developed in the vascular system of the corn plants in Field 3 during the 2010 growing season would not have been in the soil, as the plant residue would not have had time to decompose (Pudasaini,

2006). This would not have been a factor in Field 4 since a stalk shredder was used to facilitate more rapid breakdown of plant residue. Higher numbers of propagules are often associated with larger quanities of root tissue of the current crop. In corn and potatoes, the largest amount of root mass is in the top 45 cm (Hilfiker, 1988; Lesczynski, 1976). Our sampling did not exced the rooting depth to deterimine if V. dahliae populations decreased below that depth.

Conclusions

P. penetrans population densities were consistantly highest in the upper 30 cm of soil. In contrast, P. pachydermus population densities were significanly higher at the lowest depth sampled, 30-45 cm. All P. pachydermus recovered were below the 30 cm soil depth. There was no statistically significant diffrences in the distribution profile of Verticillium dahliae in relation to soil depth in either of the two fields sampled. The P. pachydermus data were consistant with previous research (Boag, 1987; Brodie, 1976; McSorley, 1990). P. penetrans is know to search out and infect crop roots in the soil profile. The precence of the majority of the P. penetrans population was detected at the depth at which the root zone of the two crops commonly grown on these fields would develope. The data calls into question the accuracy of current sampling methods. We do not know if we are getting an accurate enough view of the numbers of this nematode in the root zone. It is common practice for growers to only sample down to 20 cm

33 soil depth. Our research shows that the largest proportion of P. penetrans is in the upper 30 cm.

Paratrichodorus pachydermus is very commonly found in the 10-40 cm soil depth range.

Due to the fact that it can vector TRV, a consciencions effort needs to be taken to sample at a deeper depth than the current protocol calls for or in the spring of the year, to determine if it is in fact present at threshold levels.

Verticillium dahliae densities were not significantly diffrent from one depth to the other.

This does not, however, take into account the release of propagules from degrading plant residue in the soil. Further work needs to be done to determine the critical period of propagule release from degrading plant residue, and how deep sampling for P. penetrans should be to maximize efficiency and accuracy from each sample. This would allow potato growers to have a better idea of what their risk of potato early-die would be in the upcoming growing season.

Improvemetns in sampling for soil nematodes and other soil borne pathogens will result in reduced costs and crop losses and improved crop quality.

Chapter III

Corky Ringspot Disease of Potato Fumigant and Non-fumigant Nematicide control

Introduction

Corky Ringspot Disease of Potato (CRSD) was first detected in Michigan in 2007 (Kirk,

2008). There are currently two fields known to have this infectious disease. CRSD is caused by the virus Tobacco Rattle virus (TRV). This virus is vectored by the stubby-root nematodes

Paratrichodorus, and Trichodorus (Sol, 1960; Taylor C. B., 1997; Walkinshaw, 1961; Weingarter,

2001). There are three Trichodorus spp. and four Paratrichodorus spp. present in Michigan.

Stubby-root nematodes feed on root tips plants causing root stunting. They have a wide host range, including most crops, and common weeds (Locatelli, 1978). In most Michigan agricultural systems, unless very high levels of this vector are detected, through sampling, they are ignored. As virus vectors, however, the population density needed to cause economic damage is very low and must be managed intensely.

Tobacco rattle virus is a Tobravirus with a single strand RNA genome. It infects many different crops, including potatoes, corn and wheat (Mojtahedi, 2002). It is generally asymptomatic and does not show up until potatoes are planted in the infected field (Locatelli,

1978). It also has a very wide host range of weed species (Davis, 1975). With these host species, and its ability to survive in the vector for long periods of time, the virus is able to infest a field for up to three years without host roots present (Mac Farlane, 2003; Locatelli, 1978).

The virus particles can be lost from the nematodes body during the molting process (Mac

Farlane, 2003). Particles in the nematode virus vectors are located in the lumen, and the

35 glandular part esophagus (Taylor C. R., 1970). TRV is transmitted to the plant during the feeding process, when the stylet of the nematode is inserted into plant host epidermal cells.

Cytoplasm is removed and TRV particles are inserted by the pumping action of the esophagus

(Taylor, 1970). This virus and the CRSD have been found in many states in the U.S. It has been shown not to affect yield, but to significantly reduce the quality of the tubers (Wernette unpublished). This reduction of quality can be severe enough to cause rejection of entire potatoes by processors. Crosslin (2010) showed that the strain of TRV found in MI had a slightly different genome than other strains described in the U.S. and Europe.

Methods

Following the discovery of CRSD in Michigan, field research was initiated in 2008-2009.

The objective of the research was to determine if soil fumigation and non-fumigant nematicides could be used to reduce the vector population density enough to slow or stop infection by the

TRV. Three chemicals were used in different combinations and rates: oxymyl (Vydate C-LV), metam sodium, (Sectagon 42) and 1,3-dicloropropene (Telone II). Telone II was fall applied by

Hendrix and Dale to select plots at 198.58 kg/ha (Table 13). Metam sodium was applied in the spring of the year at 177.24 kg/ha. Oxymyl was applied in-furrow prior to planting, and at 0.56 kg/ha and 1.12 kg/ha in a band in 50 L H2O/ha and as a foliar application at tuber initiation at

1.12 kg/ha in 200 L H2O/ha and. The 16 treatments were placed in a randomized block design and replicated four times. The plots were 13.7 m wide and 50.3 m long. They were planted with certified potato seed to cv. FL 1879 on May 30th and harvested in mid October 12th.

Throughout the season, the field was managed for weeds using pre emergence herbicides as

36 well as post emergence herbicide to clean up weed seedlings. Insects including Colorado potato beetle and potato leafhopper were managed using an at plant application of a neonicotinoids insecticide as well as pyrethroid applied as a foliar when they were needed throughout the season. Fungal pathogens were managed on a seven day spray schedule using predominately chlorothalonil. These management tactics were preformed in a similar way across all fields managed by this grower.

On the 15th of October at harvest, a 12.19 m section of row from each plot was harvested. Yields were determined and 10 tubers were cut from bud end to stem end to visually determine the CRSD symptom expression. The remaining tubers were taken to the

Clarksville Research Station, and put in storage (10°C). Twice during the storage season, 50 tubers were randomly selected from each treatment and cut from bud end to stem end to determine the percent of CRSD symptom expression.

The research was repeated in 2009. A randomized block design was used with seven treatments; each replicated four times (Table 14). The plots were 10.97 m wide (12 rows) and

54.86 m long. They were planted on May 30, 2009 certified potato seed cv. FL 1879. They were harvested on the October 5, 2009. Telone II was not included due to the lack of interest from the MI potato industry. Oxymyl applications were increased in both dosage and number of applications. Oxymyl was applied at two-week intervals starting at tuber initiation and continues until a total of four foliar applications had been applied. Metam sodium was applied at the full labeled rate of 354.48 kg/ha in April of 2009, approximately four weeks prior to planting, using a shank injection applicator at a depth of 30 cm. During the season, oxymyl was

37 applied at two week intervals starting at tuber initiation and continuing until a total of four foliar applications had been applied. At harvest, tubers from four 3.05 m row sections were harvested and placed in separate crates. They were taken to the Clarksville Research Station and placed in storage (10°C). One month after harvest, tubers were removed from storage.

Tuber yields were calculated based on kg of grade A tubers per 3.05 m of row. Ten tubers were randomly selected and cut from bud end to stem end to visually inspect tubers for CRSD. This was repeated four times at four week intervals.

Tubers exhibiting visual CRSD symptoms were confirmed to have TRV using 3 methods:

1) Reverse transcription PCR RT-PCR using primers that were specific to the 16 kDa open reading frame on RNA-1 was used to confirm the presence of TRV in Dr. Kirk’s laboratory at

Michigan State University (Kirk, 2008), 2) Transmission electron microscopy to observe virus particles in tuber tissue, and 3) real-time PCR was run using similar primers as was used for the

RT-PCR at Pest Pros Inc in Plainfield, WI. (Holeva, 2006).

All plots were sampled for Paratrichodorus pachydermus before fumigation, before planting, twice during the season prior to oxymyl application and at harvest. The samples were taken at a depth of 20 cm in-row with a nematology cone shaped sampler. Approximately 20-

30 cores were taken from each plot. The samples were processed using the centrifuge floatation method (Jenkins, 1964 ). This method involves rinsing soil over a 400 mesh sieve then centrifuging the nematodes into a pellet at the bottom of a test tube. The water is then removed and a sugar solution is added to the pellet which is then mixed and centrifuged again.

The sugar nematode solution is then poured over a 400 mesh screen again and rinsed into a

38 small test tube for transport to the lab for analysis. Following extraction, the nematodes were identified down to genus, and counted using an inverted Nikon TMS stereoscope (40x).

Due to low populations of P. pachydermus (<1/100cm3 of soil), vertical distribution research was done in the fall of 2010. Three soil depths were used: 0-15 cm, 15-30 cm, and 30-

45 cm. The research was done at the two known TRV locations: Field 1 St. Joseph Co.

(41.770256N, 85.683557W) and Field 2 Saginaw Co. (43.371647N, 84.266406W). Field 1 was split into 5 sampling units. Field 2 was split into ten sampling units, as it was approximately twice as large as Field 1. One sample consisting of 20-30 probes was taken from each section.

The samples were taken with a standard agronomic soil probe (AMS, American falls ID.) with a diameter of 2.54 cm. After taking the 0-15 cm soil sample, the same hole was used to sample at soil depths of 15-30 cm and 30-45 cm. Nematodes were extracted from the samples using the centrifugal floatation method (Jenkins, 1964 ). The samples were evaluated using a Nikon

TMS inverted stereoscope (40x). All of the P. pachydermus recovered were sent to Pest Pros

Inc. for real-time PCR for TRV detection. The remaining soil was placed in black plastic 10 cm pots in the greenhouse. Pre-sprouted tobacco (Nicotiana tabacum L.) ‘SamSun NN’ was transplanted in the soil. The tobacco was monitored for eight weeks for foliar symptoms of TRV

(Holeva, 2006).

Results

There were no statistical significant difference in grade A yields among any of the 16 treatments in the 2008 research (Figure 2). The only symptom expression difference among treatments was associated with 1,3-dicloropropene + oxymyl at plant treatment (Figure 3).

Again in 2009, there were no significant difference among grade A tuber yields of the treatments (Figure 4). The control treatment had significantly higher CRSD symptom than treatments that contained oxymyl in-furrow, in combination with a foliar applications of oxymyl or treatments with spring applied metam sodium. There were no significant differences among the control and the oxymyl in-furrow alone or the foliar oxymyl alone (Figure 5).

There was statistically higher populations of P. pachydermus at all soil depths below 0-

15 cm in both fields (Table 12). The population densities were significantly higher in the 15-30 cm depth than they were in the 0-15 cm depth and the 30-45 cm depth had significantly higher population densities than both of the more shallow depths. In Field 1, 66% and 87% of the P. pachydermus, were below 30 cm in Field 1 and Field 2 respectively. The results from Pest Pros

Inc. showed no TRV in any of the P. pachydermus and no visual symptom on tobacco ‘Samsun

NN’ in the bioassay.

No TRV particles were detected by the Transmission electron microscopy. The symptomatic tubers evaluated using Reverse Transcription PCR showed negative results for TRV in most of the tubers tested. Real-Time PCR found TRV in 33% of the tubers tested, but of those positive results the amount of virus particles was at low levels.

Discussion

We found that that there was a lack of significant difference in yields in both years. This confirms other studies that report that TRV has no affect on the usable yield of the potato crop.

(Ingham, 2007). The results that were found in 2008 when we looked at symptom expression in the different treatments were hard to explain. One treatment showed significantly higher

40 symptom expression than the rest of the treatments, and was the only treatment that was significantly different than the control. This discrepancy is probably due to the variability in distribution of the nematode not only vertically in the soil profile but also horizontally across the field. When we took the 1,3-dicloropropene out of the treatments and increased the amount of applications and dosages of the metam sodium and oxymyl we saw a significant decrease in CRSD symptom expression in treatments 4) oxymyl in-furrow + four foliar application of oxymyl, 5) oxymyl in-furrow and two foliar applications of oxymyl, 6) metam sodium at 354.48 kg/ha and 7) metam sodium at 354.48 kg/ha + oxymyl applied in-furrow + four foliar applications of oxymyl. This indicates that the use of oxymyl only as an in-furrow treatment or only foliar applications was not adequate to control P. pachydermus to significantly reduce transmission of TRV.

Trichodorid nematode species are found at lower soil depths than many other nematodes (Brodie, 1976). These nematodes can be very important from a economic and product quality standpoint. They vector rod shaped virus particles that can cause yield loss and quality issues (Taylor, 1970; Hoy, 1984). The results of the vertical distribution study are similar to others that have looked at the patterns of vertical migration of Trichodorus spp. and

Paratrichodorous spp. These nematodes are migratory and prefer cool moist soils (McSorley,

1990). Studies have shown that P. pachydermus are commonly found to a soil depth of 40 cm

(Boag, 1987). Both Field 1 and Field 2 are very sandy fields with the upper strata of the fields drying out and warming up quickly leading to the vertical migration of the P. pachydermus down below a 20 cm sampling depth that was used when looking for the virus vector throughout the season.

There was no TRV detected in the 32 nematodes Pest Pros Inc tested with real-time PCR.

This was further supported by the lack of foliar symptom expression in any of the greenhouse bioassay tobacco plants. One reason that the disease might have shown up year after year while the vector found in the fall were not viruliferous. Could be that the virus particles are lost during the molt. Also due to its low threshold for infection the viruliferous nematodes might be a minute segment of the total species density thus leaving them almost undetectable. These virus particles might also have resided in weed hosts between crops and then be acquired back up by the vector just prior to infection. The results from the real-time PCR explain why no symptom expression was seen on the foliage of the tobacco in the greenhouse.

Conclusion

While there was no significant increase in yield as the amount of active ingredient of chemicals applied increased, we did observe a decrease in CRSD symptom expression in the

2009 research. This indicated that symptom expression can be decreased through the proper use of nematicides. One way to evaluate the amount of CRSD that the grower might expect in a field that has a history of CRSD is to sample for the vector. We discovered that unless the persons sampling are probing down to a depth of >30 cm, a majority of the vectors will be missed. Since only one individual can serve as a vector, it becomes difficult to determine the appropriate level to distinguish between a problem field and a field that will have only minimal infection from CRSD.

Field Zone Acres P K Mg Ca Zn CEC pH OM%

1A 2 9.5 250 194 180 900 4.9 4.4 5.9 1.5

3 12.8 220 204 140 700 4.6 3.8 5.7 2.0

4 16.4 258 186 270 1300 6.9 5.8 6.5 1.3

5 11.5 202 210 170 800 4.4 4.2 6.1 1.4

1B 1 8.7 396 264 220 1100 7.8 5.2 5.9 1.5

2 12.5 369 169 190 1000 7.1 4.7 6.2 1.0

4 6.8 374 198 260 1000 6.9 6.2 5.4 0.9

5 12.6 454 302 180 1000 9.7 4.8 5.7 1.3

7 7.7 362 142 170 900 6.2 4.3 5.7 0.8

2A 1 8.0 272 228 170 700 4.3 4.0 6.2 2.5

5 11.3 214 310 180 800 5.0 4.3 5.9 1.7

6 8.6 220 280 190 700 4.4 4.1 6.0 1.5

2B 3 9.5 322 174 170 900 5.6 4.4 5.9 0.8

Table 1. (cont’d)

4 10.0 336 226 160 1000 5.7 4.7 6.0 1.5

5 6.5 352 214 170 1300 5.4 5.4 5.9 1.1

Table 2. Phosphatase, Tyrosine aminopeptidase, N-acetyl glucosaminidase and β-1,4- glucosidase (nmol/h/g) activity associated with two potato management systems in Mecosta County, MI, March 2009.

1 Enzyme System Mean (Range) nmol/h/g S.E. p Value

Phosphatase Continuous alfalfa 151.9 (177.8-132.3) ±6.07 0.003 Potato/corn rotation 117.6 (143.5-95.8) ±7.10

Tyrosine Continuous alfalfa 8.0 (10.7-4.9) ±0.72 0.024 aminopeptidase Potato/corn 5.9 (7.2-3.8) ±0.36 rotation

N-acetyl Continuous alfalfa 16.7 (23.6-10.3) ±1.63 0.068 glucosaminidase Potato/corn 12.9 (17.8-10.5) ±0.80 rotation

β-1,4-glucosidase Continuous alfalfa 54.8 (77.9-40.2) ±4.27 0.46 Potato/corn 50.7 (66.5-41.8) ±3.19 rotation

1 Ten years of continuous alfalfa before planting to potatoes in 2009 or potato corn rotation every other year, 1999-2009.

Table 3. Phosphatase, Tyrosine aminopeptidase, N-acetyl glucosaminidase and β-1,4- glucosidase (nmol/h/g) activity associated with two soil types in Mecosta County, MI, March 2009.

Enzyme Soil Type Mean (Range) nmol/h/g S.E. P Value

N-acetyl Mecosta Sand 12.0 (14.72-10.33) ±0.60 0.014 glucosaminidase Covert Sand 15.0 (17.78-12.54) ±0.82

Phosphatase Mecosta Sand 122.6 (153.48-95.81) ±9.12 0.095 Covert Sand 149.7 (198.73-101.68) ±11.66

Tyrosine Mecosta Sand 6.3 (10.67-3.78) ±0.82 0.235 aminopeptidase Covert Sand 7.8 (11.84-5.72) ±0.87

β-1,4-glucosidase Mecosta Sand 50.8 (66.52-41.78) ±3.86 0.961 Covert Sand 51.0 (60.63-40.24) ±2.83

Table 4. Total and mean daily carbon mineralization (μg C/g soil) associated with two management systems and two soil types in Mecosta County MI, March 2009.

Carbon Mineralization System/ Soil Type Mean (Range) μg C/g soil S.E. P Value

Total

Continuous alfalfa 46.8 (65.07-30.61) ±4.22 Potato/corn 0.029 34.3 (44.92-25.15) ±2.10 rotation

Mecosta Sand 38.9 (65.07-30.62) ±4.73 0.868 Covert Sand 39.2 (53.46-25.15) ±4.19 Daily

Continuous alfalfa 2.1 (2.96-1.39) ±0.19 Potato/corn 0.029 1.6 (2.04-1.14) ±0.10 rotation

Mecosta Sand 1.8 (2.96-1.39) ±0.19 0.866 Covert Sand 1.8 (2.43-1.14) ±0.21

Table 5. Absolute nematode population density (nematodes/100 cm3 soil) associated with two management systems and two soil types in Mecosta County MI, March 2009.

System/ Soil Type1 Mean (Range) 100 cm3 S.E. P Value

Continuous alfalfa 320.7 (541-112) ±27.20 0.017 Potato/corn rotation 229.0 (367-107) ±24.06

Mecosta Sand 264.4 (508-112) ±34.96 0.364 Covert Sand 306.6 (541-107) ±29.07

1 Ten years of continuous alfalfa before planting to potatoes in 2009 or potato corn rotation every other year, 1999-2009.

Table 6. Absolute nematode population density (nematodes/100cm3 soil) of two management systems and five trophic groups and one genus in Mecosta County, MI March 2009

1 3 Trophic group System Mean (Range) 100 cm S.E. P Value

Bacterivores Continuous alfalfa 273.7 (494-143) ±24.96 Potato/corn 0.061 203.3 (354-79) ±26.30 rotation

Carnivores Continuous alfalfa 8.4 (28-0) ±1.64 0.636 Potato/corn 6.6 (48-0) ±0.51 rotation

Fungivores Continuous alfalfa 0.3 (4-0) ±0.21 Potato/corn 0.341 0.1 (1-0) ±0.07 rotation

Omnivores Continuous alfalfa 23.9 (44-12) ±1.99 Potato/corn 0.000 9.0 (21-0) ±1.85 rotation

Herbivores Continuous alfalfa 28.8 (50-8) ±2.62 Potato/corn 0.000 9.1 (32-0) ±3.03 rotation

Pratylenchus Continuous alfalfa 26.8 (46-7) ±2.50 penetrans Potato/corn 0.005 9.1 (29-0) ±2.99 rotation

1 Ten years of continuous alfalfa before planting to potatoes or potato corn rotation every two years.

Table 7. Absolute nematode population density (nematodes/100 cm3 soil) associated with two soil types and five tropic groups in Mecosta County MI March 2009.

3 Trophic group Soil Type Mean (Range) 100 cm S.E. P Value

Bacterivores Mecosta 0.166 207.6 (444-79) ±34.22 Sand Covert Sand 270.5 (494-103) ±27.43

Carnivores Mecosta 0.195 10.7 (37-4) ±4.09 Sand Covert Sand 4.8 (17-0) ±1.42

Fungivores Mecosta 0.520 0.2 (2-0) ±0.18 Sand Covert Sand 0.1 (1-0) ±0.06

Omnivores Mecosta 0.105 19.6 (37-4) ±2.51 Sand Covert Sand 13.6 (36-0) ±2.48

Herbivores Mecosta 0.107 26.3 (44-12) ±2.69 Sand Covert Sand 17.6 (5-0) ±4.42

Pratylenchus Mecosta 25.5 (12-44) ±2.60 penetrans Sand 0.068 Covert Sand 16.3 (0-46) ±4.03

Location1 Soil Type Slope Drainage Soil Profile Soil Texture (%)

Field 1 Spinks loamy 0-6 Well drained 0-25 cm Loamy sand 09’ potato, sand 25-66 cm Loamy sand 10’ corn and 66-150 cm Sand clover

Field 2 Pipestone 0-3 Somewhat poorly 0-108 cm Sand 09’ corn, sand loamy drained 10’ potato substratum

Field 3 Perrin loamy 0-4 Moderately well 0-51 cm Loamy sand 09’ corn, sand drained 51-81 cm Fine Sandy 10’ corn 81-152 cm Loam Gravelly Sand

Gilford Sandy Very poorly drained 0-28 cm Sandy loam loam 28-74 cm Sandy loam 74-152 cm Gravelly sand

Field 4 Coloma Sand 0-6 Excessively drained 0-152 cm Sand 09’ potato, 10’ seed corn

1 in St. Joseph Co. (41.770256N, 85.683557W), Field 2 in Saginaw Co. (43.371647N, 84.266406W), Field 3 in Tuscola Co. (43.530707N, 83.22839W), and Field 4 in Mecosta Co. (43.514355N, 85.365533W)

Table 9. Absolute and relative population densities associated with Pratylenchus penetrans at multiple soil depths in four Michigan fields.

Location1 Soil Depth Mean (range)2 Relative S.E. p Value (cm) Nematodes/100 cm3 density for mean

Field 1 0-15 cm 28.2 (13-49) a 35.6 ±6.24 15-30 cm 31.8 (17-46) a 40.2 ±4.81 0.269 30-45 cm 19.2 (8-35) a 24.2 ±4.91

Field 2 0-15 cm 3.8 (0-10) a 49.4 ±1.05 15-30 cm 3.1 (0-8) ab 40.3 ±0.85 0.035 30-45 cm 0.8 (0-3) b 10.4 ±0.33

Field 3 0-15 cm 20 (0-40) b 34.2 ±2.94 0.006 15-30 cm 38.5 (12-88) a 65.8 ±5.33

Field 4 0-15 cm 4.6 (0-13) b 24.3 ±0.80 0.001 15-30 cm 14.3 (4-37) a 75.7 ±2.52

1 Field 1 in St. Joseph Co. (41.770256N, 85.683557W), Field 2 in Saginaw Co. (43.371647N, 84.266406W), Field 3 in Tuscola Co. (43.530707N, 83.22839W), and Field 4 in Mecosta Co. (43.514355N, 85.365533W)

2 Column group means followed by the letters are not statistically different (P=0.05) according to Tukey’s test.

Table 10. Absolute and relative population densities of the vertical distribution of Paratrichodorus pachydermus at three soil depths associated in four potato fields in Michigan.

Location1 Soil Depth Mean (range)2 Relative S.E. (cm) Nematodes/100 cm3 density

Field 1 0-15 cm 0 (0-0) c 0.0 ±0.00 15-30 cm 2.4 (2-3) b 34.3 ±0.24 30-45 cm 4.6 (2-7) a 65.7 ±0.81

Field 2 0-15 cm 0 (0-0) c 0.0 ±0.00 15-30 cm 0.2 (0-2) b 14.3 ±0.20 30-45 cm 1.2 (0-5) a 85.7 ±0.49

Field 3 0-15 cm 0 (0-0) b 0.0 ±0.00 15-30 cm 0.75 (0-4) a 100.0 ±0.40

Field 4 0-15 cm 0 (0-0) 0.0 - 15-30 cm 0 (0-0) 0.0 -

2 Column group means followed by the letters are not statistically different (P=0.05) according to Tukey’s test.

Table 11. Verticillium dahliae propagules per gram of soil associated with two soil depths in two potato fields in MI

1 Location Soil Depth Mean (range)2 S.E. P Value (cm) Propagules/g of soil

Field 3 0-15 cm 1.6 (0-7) a ±0.43 0.386 15-30 cm 2.2 (0-6) a ±0.48

Field 4 0-15 cm 4.6 (1-20) a ±1.32 0.777 15-30 cm 5.2 (1-24) a ±1.46

1Field 3 in Tuscola Co. (43.530707N, -83.22839W), and Field 4 in Mecosta Co. (43.514355N, 85.365533W)

2 Column group means followed by the letters are not statistically different (P=0.05) according to Tukey’s test.

Table 12. Vertical distribution of Paratrichodorus. pachydermus in two fields infected with CRSD in Michigan

Location1 Depth (cm) Mean (range) Relative S.E. Nematodes/ 100 cm3 Density

Field 1 0-15 cm 0 (0-0) c2 0.0 ±0.00 15-30 cm 2.4 (2-3) b 34.3 ±0.24 30-45 cm 4.6 (2-7) a 65.7 ±0.81

Field 2 0-15 cm 0 (0-0) c 0.0 ±0.00 15-30 cm 0.2 (0-2) b 14.3 ±0.20 30-45 cm 1.2 (0-5) a 85.7 ±0.49

1 Field 1 in St. Joseph Co. (41.770256N, -85.683557W), Field 2 in Saginaw Co. (43.371647N, -84.266406W)

2 Column group means followed by the letters are not statistically different (P=0.05) according to Tukey’s test.

Table 13. 16 Chemical treatments for Corky Ringspot Disease of potato control research in White pigeon MI.

Trt Chemical Rate Trt Chemical application Rate (Kg a.i./ha) application (Kg a.i./ha) 1 Control 9 1,3-dicloropropene 198.58

2 Oxymyl at 0.56 10 1,3- 198.58 +0.56 plant D,dicloropropene+ox ymyl at plant

3 Oxymyl at 1.12 11 1,3- 198.58 +1.12 plant dicloropropene+oxy myl at plant

4 Oxymyl at 1.12 +1.12 12 1,3- 198.58 +1.12+1.12 plant+ oxymyl dicloropropene+oxy foliar myl at plant+ oxymyl foliar 5 Metam sodium 177.24 13 1,3- 198.58 +177.24 dicloropropene+Met am sodium 6 Metam sodium 177.24 +0.56 14 1,3- 198.58 +177.24 + oxymyl at dicloropropene+Met +0.56 plant am sodium+ oxymyl at plant

7 Metam sodium 177.24 +1.12 15 1,3- 198.58 +177.24 + oxymyl at dicloropropene+Met +1.12 plant am sodium+ oxymyl at plant

8 Metam sodium 177.24 +1.12 16 1,3- 198.58 +177.24 + oxymyl at +1.12 dicloropropene+Met +1.12 +1.12 plant+ oxymyl am sodium+ oxymyl foliar at plant+ oxymyl foliar

Table 14. 7 Chemical treatments for Corky Ringspot Disease of potato control research in White pigeon MI.

Trt Rate (Kg/ha) 1 Control

2 4 foliar applications Oxymyl 4.23

3 Oxymyl in furrow 2.11

4 Oxymyl in furrow + 4 foliar applications oxymyl 2.11+4.23

5 Oxymyl in furrow + 2 foliar applications oxymyl 2.11+2.11

6 Metam sodium 354.48

7 Metam sodium +Oxymyl in furrow+4 foliar applications 354.48+ 2.11+4.23 oxymyl

Field 1B (potato-seed corn rotation)

Figure 1. Four sites, Fields 1A, 1B, 2A, 2B and their associated management zones used for long-term alfalfa

vs. potato-seed corn evaluation of soil enzymes, carbon mineralization and nematode community structure in relation to soil quality. Management zones are numbered arbitrarily for organizational purposes.

SP 3,4,9 Zones

7 mile rd. SP 11, 12-14 Management Zones

3 8 1 8 8 Field 2A (continuous 2 4 7 5 alfalfa) 3 7 5 6 7 1 1

4 17 6 2 3 4 6 2 5 5 Field 1B (potato-seed corn) 9 N 9 2 4 rotation) 16 16 1 14 16 2 2 6 13 7 6 10 12 7 9 3 15 11 8 100 0 0 100 0 Feet

9 Field 1A (continuous alfalfa) SP3-4 Ma nagement Zones 09 900 0 900 Feet 1 (8.7 ac.) SP11 Management Zones 09 1 (8.0 ac .) N 80 2 (12.5 ac.) 2 (10.2 ac.) Field 2B (potato-seed corn3 (rotation)8.3 ac .) W E 3 (12.5 ac.) 4 (11.3 ac.)

5 (11.3 ac.) th 4 (6.8 ac.) 6 (8.6 ac .) S SP12-14 Management Zones 09 5 (12.6 ac.)

1 (8.4 ac .) Ave. 2 (14.3 ac.) 6 (4.4 ac.) Date: May 2, 2009 3 (9.5 ac .) Field Name: SP 12-14; 02 4 (10.0 ac.) 7 (7.7 ac.) 5 (6.5 ac .) N Location: Mecosta C o., Mic higan, U.S. 6 (7.0 ac .) 8 (6.0 ac.)

Farm Name: S P Home 7 (5.3 ac .) 8 (10.7 ac.) 9 (7.3 ac.) W E Client N ame: S ackett Potatoes 9 (6.7 ac .) Total A cres: 140.96 10 (8.2 ac .) SP9 Manageme nt Zones 09 11 (5.3 ac .) S Field Boundary S tart Location: 12 (16.7 ac.) 1 (8.3 ac.) Latitude: 43.56515925 13 (9.6 ac .) 2 (9.5 ac.) Longitude: -85.24754388 14 (10.2 ac.) 15 (4.7 ac .) 3 (12.8 ac.) 16 (4.1 ac .) Date: May 2, 2009 17 (3.8 ac .) Field Name: SP 2-4, 30; 02 4 (16.4 ac.) Location: Mecosta C o., Mic higan, U.S. 5 (11.5 ac.) Farm Name: S P Home Client N ame: S ackett Potatoes Total A cres: 154.75 58 Field Boundary S tart Location: Latitude: 43.56863740 Longitude: -85.23481490 Figure 2. Potato tuber yields associated with 16 soil fumigant and non-fumigant nematicide treatments at White Pigeon MI in 2008.1

600 2 A A A A 500 A A A A A A A A A A A A 400 300

200 Yield (cwt/A) Yield 100 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Treatments

1 Fumigant and non-fumigant nematicide treatments for the control of Paratrichodorus. pachydermus

2Yield (cwt/A) labeled with different letters are significantly different (P<0.05)

Figure 3. Corky Ringspot Disease of potato tuber symptoms associated with soil fumigant and non-fumigant nematicides treatments in White Pigeon MI in 2008.1,2

10 A2 9

5 B 4 B 3 B B

% symptom expression symptom % B B B 2 B 1 B B B B B B B 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Treatments

12008 nematicide treatments for the control of Paratrichodorus. pachydermus

2 % symptom expression labeled with different letters are significantly different (P<0.05)

Figure 4. 2009 Grade A potato tuber yields associated with nematode control for seven treatments.1,2

500 A A A2 A A A A 450

) 400 350 300 250 200

150 rade A Yield (Cwt/A A Yield rade G 100 50 0 1 2 3 4 5 6 7 Treatment

1 2009 fumigant and non-fumigant nematicide treatments for the control of Paratrichodorus pachydermus

2 Yield (cwt/A) labeled with different letters are significantly different (P<0.05)

Figure 5. 2009 TRV symptom expressions associated with seven chemical treatments1,2

3 A 2.5 AB 2

1.5 AB 1

% Symptom Expression Symptom % 0.5 B B B B 0 1 2 3 4 5 6 7 Treatments

1 2009 fumigant and non-fumigant nematicide treatments used for control of Paratricodorus. pachydermus

2 % symptom expression labeled with different letters are significantly different (P<0.05)

Figure 6. A-D. Possible tobacco rattle virus crystal structures seen in symptomatic tubers photos taken under Transmission Electron Microscopy

A B

C D

APPENDIX

Soil Fumigation Guide

Soil fumigants are used commonly in Michigan potato production to decrease the yield losses caused by soil-borne pathogens such as the Verticillium-wilt fungus (Verticillium dahliae) and penetrans root-lesion nematode (Pratylenchus penetrans). Recently, the Environmental protection agency (EPA) has completed the process of re-registration of the soil fumigants as mandated by the Federal Insecticide, Fungicide and Rodenticide Act. This includes metam sodium, chloropicrin and 1,3-D. This re-registration process results in new label requirements in further detail below. These are being released in Phase One and Phase Two label. The

Phase One label was published in December 2010. The Phase Two label will be released at the end of 2011. The purpose of their fumigation field guide is to describe the fumigation management plan, Buffer zone, post application Report and Good Agricultural practices associated with the new fumigant labels.

The following document is intended to increase awareness and act as a field reference for potato growers participating in soil fumigation for soil borne pathogen control.

Below that is an example of a field research trial looking to mediate the impact of Corky

Ringspot disease on potato production. This research was done using fumigant and non- fumigant nematicides to control the virus vector Paratrichodorus pachydermus.

Potato Fumigation Guide Figure 7. For interpretation of the references to color in this and all other figures, the reader is referred to the electronic version of this thesis. Figure 8

Figure 9

A field guide to the new fumigant labels

In Michigan Potato Production

Loren G. Wernette

Potato Fumigation Guide

A field guide for new soil fumigation labels in Michigan potato production

Potato Soil Fumigation…………………………………………………………………………………….………3

Soil fumigant re-registration process……………………………………………………………….……..3

Potato Early-Die……………………………………………………………………………………………….……..3

Early-die management History……………………………………………………………………………....4

Soil fumigants…………………………………………………….……………………………………………….….5

Fumigant management plan……………………………………………………………………………….….6

Buffer zones ………………………………………………………………………………………………………….12

Post Application Summary …………………………………………………………………………………….13

Good agriculture practices (GAP’s)…………………………………………………………………….….16

FMP and PAS Templates……………………………………………………………………………….……….21

Useful Resources………………………………………………………………………………………….……….22

CRSD Example……………………………………………………………………………………………….….…..23

References……………………………………………………………………………………………….……….….30

Soil Fumigation: Soil fumigants are used commonly in Michigan potato production to decrease the yield losses caused by soil-borne pathogens and nematodes. Recently, the Environmental Protection Agency (EPA) completed the re-registration of soil fumigants as mandated by the Federal Insecticide, Fungicide and Rodenticide Act. The re-registration process includes metam sodium, chloropicrin and 1,3-D. This re-registration process resulted in new label requirements, being released in two phases. Phase one labels were published in December 2010, and Phase Two labels in 2011. The purpose of this fumigation field guide is to describe the fumigation management plan, buffer zone, post application Report and Good Agricultural Practices associated with the new fumigant labels.

Potato Early-Die (PED) Potato Early-Die is caused by an interaction between the penetrans root-lesion nematode (Pratylenchus penetrans) and Verticillium-wilt fungus (Verticillium dahliae). It affects 50% of Michigan’s potato acreage. If not managed, this infectious disease causes tuber yield losses as high as 50%.

Root-Lesion Nematode Figure 10 Pratylenchus penetrans

Peter Mullins 2001

Figure 11 Figure 12 Root lesion nematode in plant root tissue

Corn Roots infested with root-lesion nematode

www.plantpath.wisc.edu www.apsnet.org

Figure 7, 8 Potato Early-Die Symptoms. - Infected plant + stem discoloration Figure 13 Figure 14

Early-Die Management After PED was noticed in MI potato production in the 1970’s. Shortly after beginning to use the soil fumigant 1.3-dicloropropene, aldicarb (Temik) used extensively. Aldicarb product provided excellent root-lesion nematode, insect and PED control. When aldicarb was no longer available, oxamyl, ethoprop and metam sodium were used. Initially, metam sodium was applied through center pivot irrigation systems. This changed to shank injection with the active ingredients diluted with water. Today, metam sodium is applied at 40 gallons per acre using modern spray-blade technology. Compost is also applied the fall before potato planting for soil quality enhancement.

Figure 15

Wernette, 2009

Soil Fumigants

There are three main fumigants used in MI potato production. Metam sodium is the most widely used soil fumigant in Michigan potato production Chloropicrin and Telone II are also used but on less acreage. Chloropicrin is commonly known as tear gas. It is used on a small amount of acreage in MI. 1,3-dicloropropene (Telone II) has been used in MI Potato production. All three of these fumigants are applied in similar ways generally with shank injection or spray blade.

Figure 16 Figure 17

Metam Sodium fumigation applicator

Wernette, 2009 Water being sprayed out of the Spray blade fumigator Figure 18 nozzles

Wernette, 2009

Chloropicrin Metam 1,3-D Table 15 Sodium/ Potassium

Shank injection

Spray blade

Chemigation, drip Chemigation,

sprinkler Chemigation,

center pivot

Rotor tiller Figure 19

Bio-fumigation is the process of incorporating living green tissue from certain plant families most commonly Brassica’s (yellow mustard, rape seed radish) into the soil. When incorporation takes place the plant tissue needs to be cut up into small pieces so a chemical can be released into the soil that is detrimental to the health of soil borne pathogens such as P. penetrans and V. dahliae. The results from this process can be mixed but, could be used effectively combined with other management strategies.

Fumigant Management Plan

The Fumigant Management Plan (FMP) consists of the following 14 components. A 15th section has been included to provide information about the proposed buffer zones

1. Certified Applicator: Supervising the application, the certified applicator supervising the Fumigation This section requires the recording of

contact information of the person that is in charge of the application in the field that is being fumigated. This is probably going to be whoever is running the applicator.

Figure 20 2. General site Information: Field Location County, township section and quadrant or Global Positioning system (GPS) coordinates.

www.trimble.com

Figure 21

3. Owner/Operator: the owner of the field or farm fumigation is taking place.

www.mipotato.com

4. Record keeping: record keeping is a simple check box that insures that you know that you need to keep this FMP for

two years following the fumigation in case there is an issue that come up following fumigation.

Figure 22

5. General Application Information:

 date of application  the fumigant product name  application method and rate,  depth of injection  number of acres that are being fumigated.

6. Emergency Response Plan: the emergency response plant is designed to show anyone that looks at the FMP including the handlers that are working in the field at the time of application where the evacuation routes are, where the nearest telephone is and where the contact information is available for first responders. It also includes a section on what emergency procedures are in case of an incident such as equipment failure: complaints or if there is elevated air concentrations of the fumigant. This section should be typed and attached to the FMP. It should include a map or diagram of the field and evacuation routes.

Table 16 Trigger Requirement Distances; triggered the applicator must a) 18 monitor the site or b) provide information to neighbors about the fumigation application.

If the buffer zone is AND occupied structures are within _____ from the edge of the ______: buffer zone,

> 25 feet and ≤ to 100 feet 50 feet

> 100 feet and ≤ 200 feet 100 feet

> 200 feet and ≤ 300 feet 200 feet

> 300 feet 300 feet

7. Communications: Communications between the applicator and owner/operator and other on-site handlers

requires that a copy of the fumigant label and MSDS sheet are present at the site of application for employee review. It also indicates that the certified applicator will be at the application during all fumigant handling. If they will not, then there needs to be a plan on how the certified applicator will share the label requirements with the handlers and owner/operator. It also requires an indication of who will be at the fumigation site after application until the entry restricted period expires.

Table 17 Product Minimun Minimum number of Minimum formulation number air-purifying number of of respirators SCBAs handlers

chloropicrin 2 2 Full-face 1 Metam sodium 1 1 Full-face 0 Metam potassium 1 1 Full-face 0

Figure 23

8. Handler Information: Information for all handlers must be attached to the FMP. A template for this is provided at http://www.epa.gov/oppsrrd1/reregistration/soil _fumigants/

Figure 24

9. Tarps: Tarps are used to control off gassing of the fumigant. Almost no tarping is done with metam sodium but if methyl bromide is being applied, tarping is essential.

Figure 25

Figure 26

10. Soil: Soil texture, organic matter temperature and moisture data are required, including on indication of how soil moisture was determined and what method was used to determine soil moisture.

Figure 27

11. Weather: Weather conditions play a large role in how likely issues with off gassing and fumigant efficacy will arise. A summary of the weather data for the day of application and for the 48 hours following application should be included. These data can be obtained on-line at www.nws.noaa.gov

www.agroweather.geo.ms

u.edu

Figure 28

12. Posting: The individual responsible for posting the fumigation treatment area should be identified, as well as the date the signs are to go up and when they should be taken down.

13. Air Monitoring: The air monitoring plan indicates Figure 29 how the applicator is going to monitor off-gassing and if off-gassing leading to irritation what steps will be taken. If irritation is detected, the applicator is required to either stop fumigation or everyone associated with the fumigation application must wear respiratory protection the remainder of the application. This section must also include a full face respirator response plan that states if a handler experiences any sensory irritation when wearing a full face respirator or a MITC air sample is taken and the number exceeds 6000 ppm, the application must stop or all handlers must be removed Figure 30 from the fumigation area. At this point, the emergency response plan would be activated.

14. Good Agricultural Practices: A copy of the label can be attached and the GAP’s to be included can be highlighted. If this is not done, then the application method boxes on the FMP template must be marked. The GAP’s for spray blade and shank injection include wind speed >2 mph at the start of the application or projected to reach at least 5 mph during application. Weather conditions should be monitored. If a temperature inversion is forecasted to persist for 18 consecutive hours during the 48 hours following application. The fumigation should be postponed. Soil conditions include soil texture and organic matter.

Injection depth and type of soil sealing should follow label requirements. Soil moisture needs

to be between 60-80% of field holding capacity. Soil Temperature needs to be lower than 90°F. Application and Equipment should not be in disrepair and should follow the practices described in the label. 77

Buffer Zone: buffer zones will be included in the Phase 2 Label. Buffer zone signs must include:

 Do not walk Symbol.  DO NOT ENTER/NO ENTRE.

 [Name of Fumigant Name of Product]. Fumigant Buffer Zone.  Certified Applicator information

Table 18 % Reduction Condition MeBr Chloropicrin Metam Dazomet Use of h igh b arrier t arp 30 or 60 30 or 60 15 or 30 NA Organic > 1% - 2% 10 10 10 10 matter content > 2% - 3% 20 20 20 20 > 3% 30 30 30 30 Clay c ontent > 27% 10 10 10 10 Soil Temp ? 50 F (center NA 10 20 NA pivot and shank) > 50 - 70 F (center NA NA 10 NA pivot and shank) > 50 - 70 F ( drip irrigation, flood irrigation, and NA NA 10 NA rotary tiller and spray blade applications ) Symmetry application system, high NA 40 or 70 NA NA barrier tarp, <100 lb ai/A Potasium t hiosulfate seal with 15 15 NA NA water applied over tarp Water seal applied over tarp NA 10 NA NA Max reduction 80 80 80 40

Post Application Summary

Following application, a post application summary must be completed. This documents any differences that might have occurred in the application that was not foreseen in the FMP. As well as document any occurrence of irritation due to the fumigant or complaint from a pedestrian or bystander. Many of these sections are the same in this document as they are in the FMP and if no deviations from the original plan occurred they have check boxes that indicate this lack of difference. The sections in the post application summary are as follows.

General Application Information: This section is the same as the general application information section on the FMP and is meant to be Text Boxes 26-35 used to provide basic information about the fumigant application in regards to how and when the fumigant was applied.

Figure 31

Weather: This section also mirrors a section on the FMP. If any weather not represented by the weather forecast on the FMP, weather report should be attached.

Figure 32

Tarp Damage and Repair, this section is only applicable to operations that use tarps as part of their fumigation application.

Tarp Perforation/Removal, when were the tarps perforated or removed and how were they removed. Not applicable if no tarps were used.

Complaints: This section records complaints from individuals on-site or off site. If the person was off-site include their name, address and phone number. It should

also provide space for comments on what additional control measures and emergency procedures were performed following the complaint, and any additional comments that

are applicable to the report.

Figure 33 Incidents: Any incident that happened during fumigation should describe why the incident happened. What emergency procedures were followed, the date and time of the incident if state agency was notified.

Wernette, 2009

Communications: This is a follow up to the communication section of the FMP in regards to the presence of the applicator at the application site during all handler activities that took place from the beginning

of fumigation to the end of the 48 hour entry restricted period. If the certified applicator was not on-site for all handler activities the names and phone numbers of the persons contacted should be listed.

Figure 34

Posting: Dates of fumigant treated area and buffer zone sign removal should be recorded.

Figure 35

Handler Changes: If there any changes in the handler information that were on-site that were not on the FMP they should be to attached to the post application summary.

Figure 3 6

Other Deviations: not accounted for in the original FMP need to be recorded.

Good Agricultural Practice

Good Agricultural Practices are described on the new labels for the soil fumigants. These describe the best practices to increase the efficacy of the fumigant as well as limit the amount of off-gassing that will occur during an application.

Figure 37 Soil Tilth: Several conditions must exist for fumigation of metam sodium with spray blade shank injection. First of all soil preparation is very important to the efficacy of the fumigant. The soils must be in good tilth and free of large clods if subsurface hard pans are present in the area of desired fumigation deep tillage should be used to fracture these layers. Plant residue that is present must not interfere with the application of the soil seal which is used to limit natural chimney’s that facilitate the rapid loss of volatilized fulminate into Figure 38 the atmosphere. Wind Speed: Wind speed must be at least 2 mph or forecasted to reach 5 mph during application. The wind speed forecast for the day of the application and 48 following application should be monitored. If weather conditions are not optimal, fumigation should not proceed. You should not apply if a temperature inversion is forecasted to persist

for more than 18 consecutive hours for the 48 hour period after application.

Soil temperature: during application is extremely

important if the soil temperature is not in the ideal range

there will be lose of efficacy of the fumigant. The soil temperature at the beginning of the application should not be above 90°F. If the air temp has been above 100°F in any

of the three days prior to application, soil temperature must be measured and recorded in the FMP.

Unfavorable weather: Monitoring of air inversions which block the upward movement of air trapping fumigant vapors near the ground. These

resulting vapors can move unpredictably to off-site locations. Detailed weather condition may be obtained on-line at www.nws.noaa.gov, or by

contacting your local national weather service forecasting office.

Soil moisture: Soil moisture in the top six inches must be between 60% and 80% of field capacity prior to application. If soil moisture potential measuring equipment is not used, the USDA Feel Method may be used. If there is insufficient moisture throughout the top

six inches of soil immediately prior to the application the soil moisture can be adjusted. If there is adequate soil moisture below six inches tillage can be used during or immediately prior to application.

Table 19 USDA Feel Method

Soil Texture Soil Ball structure at 60-80% moisture Coarse Texture weak ball with loose and clustered sand grains on fingers darkened color moderate water staining on fingers will not ribbon

Moderately coarse ball with defined finger marks very light soil water staining on Texture fingers darkened color will not stick

Medium Textured ball and will leave a very light staining on fingers and will form a weak ribbon between the thumb and forefingers

Fine Texture ball with defined finger marks light soil water staining on fingers ribbons between thumb and forefinger

If more than one soil texture should be consider the lightest soil texture that must comply with the soil moisture requirements. The field can be divided into areas of similar soil texture and soil moisture of each area should be adjusted as needed. If soil moisture is high the effect of the fumigant will be retarded and effectiveness of the treatment will be reduced.

Figure 39

Following application chisel traces must be eliminated following an application using one or more of the following methods:

Figure 40

Compaction with bed shaper, roller, press wheel or similar device and/or Covering the treated soil with 3-6 inches of untreated soil using tillage equipment.

Figure 41 Wernette, 2009

Applying a minimum of ½ inch of water beginning immediately after application of a set and completing the water treatment with four hours.

www.vallygreeley.vallydealers.com

Certain practices should be avoided under the

GAP’s these include and are not limited to

Figure 42 Do not apply or allow fumigant to drain or drip onto the soil surface All tanks hoses fittings valves and connections must be serviceable tightened, sealed and not leaking Dry connect fittings must be installed on all tanks and transfer hoses. Wernette, 2009

Figure 43

Application equipment, Sight gauges, and pressure gauges must be working. Nozzles and metering devices must be the correct size and sealed and unobstructed each nozzle must be equipped with flow monitor. For undiluted product aluminum brass copper galvanized iron and zinc materials cannot be used. Wernette, 2009

Figure 44

Wernette, 2009

 All applicators must include a filter to remove and particulate from the fumigant and a check valve that is visible to the tractor pilot during application to prevent backflow of the fumigant into the pressurized cylinder o Before using a fumigation rig for the first time of when preparing it for use after storage the operator must check the following items carefully o CheckSoil the Fumigation filter and clean Management or replace eth Plan filter Templateelement as required o Check all tubes and chisels to make sure they are free of debris and (seeobstructions http://www.epa.gov/oppsrrd1/reregistration/soil_fumigants/ ) o Check and clean orifice plates

Soil Fumigation Post Application Summary Report Template

(see http://www.epa.gov/oppsrrd1/reregistration/soil_fumigants/)

Useful Resources http://www.epa.gov/oppsrrd1/reregistration/soil_fumigants/ www.nws.noaa.gov, www.pestid.msu.edu

References

Baldridge, G D, O’Neill N R, Samac D A. “Alfalfa (Medicago sativa L.) resistance to the root-lesion nematode, Pratylenchus penetrans: defence-response gene mRNA and isoflavonoid phytoalexin levels in roots.” Plant Molecular Biology 38 (1998): 999-1010.

Bell, NL, Watson, RN. "Dynamics of sympatric Paratylenchus nauns and Paratrichodorus minor populations in soil under pasture." Nematology 3, no. 3 (2001): 267-275.

Bird, G W. "Influence of Incubation Solution on the Rate of Recovery of Pratylenchus brachyurus from Cotton Roots." Journal of Nematology, 1971: 3:378-385.

Boag, B, Brown D J. F., Topham, P B. "Vertical and Horizontal Distribution on Virus-Vector Nematodes and Implications for sampling procedures." Nematology, 1987: 83-96.

Brown, D.J.F., Robertson, W.M., Trudgill, D.L. "Transmission of Viruses by Plant Nematodes." annul Review of Phytopathology, 1995: 33:223-49.

Cochran, G, Snedecor W, William G. Statistical Methods sixth edition. Ames, Iowa: The Iowa State University press, 1937.

Crosslin, J.M. Hamm, P.B., Kirk, W.W.,Hammond, R. W. "Complete genomic sequence of Tobacco rattle virus isolate from Michigan-grown potatoes." Arch Virology 155 (2010): 621-625.

Davis, J.R., Allen, T.C. "Weed Host of Tobacco Rattle Virus in Idaho." American Journal of Potato Research 52, no. 1 (1975): 1-8.

Fernandez-Calvina D., Soler,R. P., Polo, A., Diaz, M., Arias, E. M., Plaza, C. "Enzyme activities in Vinyard Soil Long-Term Treated with copper-based Fungicides." Soil Biology and Biochemistry, 2010: 1-9.

Forge, T.A., DeYoung R., Vrain T. C. "Temporal Changes in the Vertical Distribution of Pratylenchus penetrans under Raspberry." Journal of Nematology 30, no. 2 (1998): 179-183.

Gallo M., Amonette R., Lauber C., Sinsabaugh R.L., Zak D.R. "Microbial Community Structure and Oxidative Enzyme Activity in Nitrogen-Amended North Temperate Forest Soils." Microbial Ecology, 48, 2004: 218-229.

Goodell, P.B. Ferris, H. "Sample Optimization for Five Plant-Parasitic Nematodes in an Alfala Field." Journal of Nematology, 1981: v.13; 304-313.

Grandy AS, Robertson GP. "Land-Use intesity Effects on soil Organic Carbon Accumulation Rates and mechanisms." Ecosystems, 2007 : V. 10: 58-73.

Grandy, A.S., Strickland, M. S., Lauber, C. L., Bradford, M.A., Fierer, N. "The influence of microbial communities, managment and soil texture on soil organic matter chemistry." Geoderma, 2009: 150:278- 286.

Haase, S., Philippot, L., Neumann, G., Marhan, S., Kandeler, E. "Local response of Bacterial Densities and

Enzyme activities to Elevated Atmospheric CO2 and different N Supply in the Rhiczosphere of Phaseolus vulgaris L." Soil Biology and Biochemistry, 2008: 40:1225-1234.

Haynes RJ, Beare MH. "Affregation and organic matter storage in meso-thermal, humid agricultureal soils." In Structure and organic matter storage in Agricultureal soils., by Stuart BA Carter MR, 213-62. New York: CRC Press, 1996.

Hilfiker, RE, Lowery, B. "Effects of Conservation Tillage Systems on Corn Root Growth." Soil and tillage Reserach 12 (1988): 269-283.

Holeva, R., Phillips, M.S., Neilson, R., Brown, D.J.F., Young, V., Boutsika, K., Blok, V.C. "Real-Time PCR detection and quantification of vector trichodorid nematods and Tobacco rattle virus." Molecular and Cellular Probes 20 (2006): 203-211.

Hoy, JW, Mircetish, SM, Lownsbery, BF. "Differential Transmission of Prunus tomato Ringspot Virus strains by Xiphinema califonicum." Ecology and Epidemiology, 1984: 332-335.

Ingham, R.E., Hamm, P.B., Baune, M., Merrifield, K.J. "Control of Paratrichodorus allius and Corky Ringspot Disease of Potato with Shank-injected Metam Sodium." Journal of Nematology 39, no. 3 (2007): 258-262.

Jansa, J. "Soil tillage affects the community structure of mycorrhizal fungi in maize roots." Applied Ecology, 2003: V. 13 1164-1176.

Jenkins, W.R. "A rapid centrifugal-floatation technique for seperating nematodes from soil." Plant Disease, 1964 : 48:692.

Johnson, S. B. Verticillium Wilt of Potatoes. bullelin #5041, Orono: University of Maine Cooperative Extension.

Jones, FGW, Larbey D W, Parrott, D M. "The influence of soil structure and moisture on nematodes especially Xiphinema, Longidorus, Trichodorus and Heterodera spp." Soil Biology and Biochemistry, 1969: 1: 153-169.

Kable P. F., Mai W. F. "Influence of Soil Moisture on Pratylenchus penetrans." Nematologica, no. 14 (1968): 101-122.

Kirk, W.W., Gieck, S.L., Crosslin, J.M., Hamm, P.B. "First report of Corky Ringspot caused by Tobacco rattle virus on Potato (Solanum tuberosum) in Michigan." Plant Disease 92 (2008): 485.

Koch O., D. Tscherko, E. Kandeler. "Temperature sensitivity of Microbial Respiration, Nitrogen Mineralization and Potential Soil Enzyme Activities in Organic Alpine Soils." Global Biogeochemical cycles, 2007.

Lammirato, C., Miltner, A.,Wick, L., Kastner, M. "Hydrolysis of Celloboise by B-glucosidase in the Presence of Soil Minerals-Interactions at Solid Liquid Interfaces and Effects on Enzyme Activity Levels." Soil Biology and Biochemistry, 2010: 1-8.

Lesczynski, DB, Tanner, CB. "Seasonal Variation of Root Distribution of Irrigated, Field- Grown russet Burbank Potato." American Journal of Potato Research 53 (1976): 69-78.

Locatelli, E. A., Allen, T.C., Koepsell, P.A., Appleby, A.P. "Diagnosis of Tobacco Rattle Virus (TRV) and other Viruses in Weed and Rotation Crops in potato fields." American Journal of Potato Research 55, no. 5 (1978): 249-257.

Mac Farlane, S.A. "Molecular determinats of the transmission of plant viruses by nematodes." Molecular Plant Pathology 4 (2003): 211-215.

Madejon, E., Burgos, P., Lopez, R., Cabrera, F. "Soil Enzymatic Resposnce to Additions of Heavy Metals with Organic Residues." Biology and Fertiity of Soils, 2001: 144-150.

McSorley R., Dickson D.W. "Vertical Distribution of Plant-parasitic Nematodes in Sandy Soil under Soybean." Journal of Nematology 22, no. 1 (1990): 90-96.

Mojtahedi, H, Crosslin, J.M., Thomas, P.E., Santo, G.S., Brown, C.R.,Wilson, J.H. "Impact of Wheat and Corn and Rotational Crops on Corky Ringspot Disease of Russet Norkotah Potato." American Journal of Potato Research 79 (2002): 339-344.

Muruganandam S., Israel D.W., Robarge W.P. "Activities of Nitrogen-Mineralization Enzymes Associated with Soil Aggregate Size Fractions of Three Tillage Systems." Soil Biology and Biochemistry, 2009: 751- 759.

NASS. Crop Production 2010 Summary. Annual Report, USDA National Agricultural Statistics Service, 2011.

Nicot, P C, Rouse, D I. "Precision and bias of three quantitative soil assays for Verticillium dahliae." Phytopathology, 1987: 77: 875-881.

Pudasaini, M. P., Schomaker C. H., Been T.H., Moens M. "Vertical Distribution of plant-parasitic nematode Pratylenchus penetrans under four field crops." Nematology 96, no. 3 (2006): 226-233.

Rangahau, M. K. Potato early dying (Verticillium wilt). Extension Bulletin, Lincoln: New Sealand Institute for Crop and Food Reseach. Rasse, D.P Smucker, A.J.M. "Root recolonization of previous root channels in corn and alfalfa rotations." Plant and Soil Science, 1998: v. 204; 203-212.

Robertson GP, Grandy AS. "Soil System Managment in Temperate Regions." In Biological Approaches to Sustainable Soil Systems, by Ball AS, Fernandes E, Herren H, Husson O, Laing M, Palm C, Pretty J, Sanchez P, Sanging N, Thies J. Uphoff N, 27-39. Boca Raton: CRC Press, 2006.

Robertson GP, Wedin D, Groffman BM, Blair JM, Holland EA, Nadelhoffer KJ, Harris D. "Soil Carbon and Nitrogen Avaliability: nitrogen minerilization nitrification and soil respiration potentials." In Standard Soil Methods for Long Term Ecological Research, by Coleman DC, Bledsoe CS, Sollins P, Robertson GP, 258- 71. New York: Oxford University Press, 1999.

Robertson, PG, Freckman, DW. "The spatial distribution of nematode trophic groups across a cultivated ecosystem." Ecology 76, no. 5 (1995): 1425-1432.

Rowe, R.C. Riedel, R.M. Martin, M.J. "Synergistis Interactions Between Verticillium dahliae and Pratylenchus penetrans in Potato Early Dying Disease." Phytopathology, 1985: v.75 412-418.

Saiya-Cork, KR Sinsabaugh, RL Zak, DR. "The effects of long term nitrogen deposition on extracellular enzyme activity in an Acer Sachharun forest soil. ." Soil Biology Biochemistry 34, 2002: 1309-1315.

Sarapatka, B. Phosphatase actifities in agroecosystem soils. Uppsala: Swedish University of Agricultural Sciences, 2003.

Sol, H.H., Van Heaven, J.C., Seinhorst, J.W. "Transmission of rattle virus and Atropa belladonna mosaic virus by nematodes." Tijsahrift Over Plantenziekten 66 (1960): 228-231.

Stevenson, F.J. Humus chemistry:Genesis, composition, reaction. New York: John Wiley & Sons, 1994.

Taylor, C E, Robertson, W M. "Location of Tobacco Rattle Virus in the Nematode Vector, Trichodorus pachydermus Seinhorst." Journal of general Virology 6 (1970): 179-182.

Taylor, C.E., Brown, D.J.F. Nematode vectors of Plant viruses. Wallingford, UK.: CAB international, 1997.

Taylor, R.J., Pasche, J.S., Gudmestad, N.C. "Influence of Tillage and method of Metam Sodium Application on Distribution and Survival of Verticillium dahliae in the Soil and the Development of Verticillium Wilt of Potato." American Journal of Potato Research, 2005: 82:451-461.

USDA. "Custom Soil Resource Report for Tuscola County Michigan." Web Soil Survey. Febuary 7, 2011. websoilsurvey.nrcs.usda.gov/app/websoilsurvey.aspx (accessed Febuary 7, 2011).

W., Shi. "Agricultural and Ecological Significance of Soil Enzymes: Soil Carbon Sequestration and Nutrient Cycling." In Soil Enzymology, by Varma A Shukla G, 43-60. Verlag Berlin Heidelberg: Springer, 2011.

Walkinshaw, C.H., Griffin, G. D., Lawson, R.H. "Trichodorus christei as a vestor of Potato Corky Ringspot (tobacco rattle) virus." Phytopathology 51 (1961): 806-808.

Weingarter, D.P. "Potato Viruses with soil-borne vectors." In Virus and Virus like dieseae of potatoes and Production of seed Potatoes, by G.,Berger, P.H., Bruat, A.A., Lawson, R.H. Loebenstein, 177-194. Boston, MA: Academic publishers, 2001.