ABSTRACT CORBETT, BRET TAYLOR. the Biology And

ABSTRACT

CORBETT, BRET TAYLOR. The Biology and Distribution of Listronotus maculicollis in Western North Carolina to Manage Turfgrass in the Southeastern U.S. (Under the direction of Rick Brandenburg). Annual bluegrass weevil (ABW), Listronotus maculicollis Kirby (Coleoptera:

Curculionidae) is a major insect pest of highly maintained turfgrass in the northeastern United

States and in some provinces of Canada. It was first observed in North Carolina in 2006 (B.T

Corbett personal communication) and has spread throughout golf courses located the mountains of North Carolina. The objectives of this research were to 1) determine the distribution of L. maculicollis in western NC and 2) to compare adult population densities between two host plants, annual bluegrass and creeping bentgrass.

The first objective sampled 46 golf courses to determine ABW presence during the summer of 2016 and 2017. Sampling was conducted in western NC where cool-season turfgrasses are the dominant turf species on golf courses. Presence of ABW in turfgrass was determined using a soap water flush (14.7ml Joy Lemon dish liquid to 3.7L water), within a one square meter area, on fairways, tees and greens. Golf courses were selected based on availability of courses with damage or previous knowledge of ABW presence. ABW was detected on 31of

46 golf courses in 2016 and 2017.

The second objective of this research project was examined utilizing field experiments conducted during the summer of 2016 and 2017, and consisted of golf courses sampled for adult

ABW in mixed stands of annual bluegrass and creeping bentgrass. The percentage of turfgrass species composition was determined using a weed grid and adult’s presence in the turfgrass areas was confirmed using a soap water flush. Data from five golf courses in 2016 and three golf courses in 2017 indicate adult ABW populations were not significantly different among areas with different concentrations of annual bluegrass encroachment into the creeping bentgrass.

ABW host preference, antibiosis and antixenosis was observed in creeping bentgrass

(Agrostis stolonifera L.), annual bluegrass (Poa annua) and hybrid bermudagrass (Cynodon dactylon L.). There were no differences in ABW survival and resistance to feeding found with any of the different turfgrasses. No eggs or larvae were found during the experiment indicating there was no oviposition or egg survival in this study.

Confirmation of pest species found in North Carolina was compared to turfgrass weevils feeding on annual bluegrass in other states. Insect samples collected were subjected to further investigation by Dr. Lourdes Chamorro, (Research Entomologist/Curator of Curculionidae c/o

Smithsonian Institution - National Museum of Natural History, Washington D.C.), Dr. Matt

Bertone (Taxonomist North Carolina State University Plant Disease and Insect Clinic, Raleigh,

NC), Dr. Terri Billeisen (Extension Specialist, Department of Entomology and Plant Pathology,

North Carolina State University, Raleigh, NC). Adult ABW were collected from North Carolina,

New Jersey, Virginia, Rhode Island, Pennsylvania, Massachusetts, Michigan, Delaware, New

Hampshire, and Canada (Toronto) for subsequent genome evaluation. Genetic sequencing

(DNA) was conducted and all specimens appear genetically identical and appear to be L. maculicollis.

The Biology and Distribution of Listronotus maculicollis in Western North Carolina to Manage Turfgrass in the Southeastern U.S.

by Bret Taylor Corbett

A thesis submitted to the Graduate Faculty of North Carolina State University in partial fulfillment of the requirements for the degree of Master of Science

Entomology

Raleigh, North Carolina

2018

APPROVED BY:

______Rick Brandenburg Travis Gannon Chair of Advisory Committee

______James Kerns

DEDICATION

To my friends and family for their constant support. To my father and mother who showed immense guidance and confidence when I needed it the most. I desire to make my loved ones proud.

ii BIOGRAPHY

Bret Taylor Corbett was born on 9 March 1993 and is the son of Jerry and Antonette

Corbett. Bret was raised in Selma, North Carolina along with his older brother Dustin Corbett.

Located in rural Johnston County, his love for the outdoors was instilled in him at an early age through his work on his family’s farm. Growing up, he played baseball competitively and golf leisurely for many years. During high school, he managed home lawns and golf courses where he developed a passion for turfgrass management. Bret graduated from North Johnston High School in 2011. He attended Lenior Community College leading him to transfer to North Carolina State

University where he earned a Bachelor of Science degree in Crop Science with a concentration in Turfgrass Science in 2015.

In January 2016, Bret began his Masters research at North Carolina State University

Department of Entomology graduate program under the direction of Dr. Rick Brandenburg, with a focus on insect control in turfgrass. His research aimed to determine the location and biology of the annual bluegrass weevil, Listronotus maculicollis, in western North Carolina golf courses.

iii ACKNOWLEDGEMENTS

This work would not have been possible without the funding from the Center for

Turfgrass Environmental Research and Education (CENTERE). I would like to first acknowledge my advisor, Dr. Rick Brandenburg, for the continuous support of my Masters research, insightful comments, for his immense patience, and for steering me in the right direction whenever I needed it. I would also like to acknowledge my committee members, Dr.

Jim Kerns and Dr. Travis Gannon whose guidance and knowledge helped me complete this research. I would like to thank our laboratory technician, Brian Royals, and extension associate,

Dr. Terri Billeisen for your insight and assistance in the lab as well as the field throughout this project.

To my family, thank you for your unconditional love and support throughout my academic career. Thank you to my parents, Jerry and Antonette, for always believing in me to follow my dreams. Thank you to my brother, Dustin for your help whenever I needed an extra hand making something for research. Thank you, Nicole and Luke, for always putting a smile on my face when I was able to come home. I hope I have made you proud.

Finally, thank you to my friends and fellow graduate students for the good times and memories shared throughout the years. Thank you to Hunter Williams and Caleb Kearney for your constant help on my project and for being great roommates. I would also like to thank

Shawnee Gundry. Graduate school would have been so boring and much more difficult without her love, support and encouragement.

iv TABLE OF CONTENTS

LIST OF TABLES ...... vii

LIST OF FIGURES ...... viii

A REVIEW OF THE LITERATURE...... 1

History, Origin, And Identification ...... 1

Current Distribution ...... 5

General Biology and Seasonality ...... 5

Host Plants ...... 8

Management ...... 10

THE PURPOSE AND FOCUS OF THIS RESEARCH ...... 14

Literature Cited ...... 15

CHAPTER 1 ...... 18

Introduction ...... 19

Materials and Methods ...... 21

Results and Discussion ...... 24

Literature Cited ...... 27

CHAPTER 2 ...... 33

Introduction ...... 34

Materials and Method ...... 36

Results and Discussion ...... 38

APPENDICES ...... 61

APPENDIX A ...... 62

v Introduction ...... 64

Material and Methods ...... 66

Results and Discussion ...... 70

APPENDIX B ...... 79

Literature Cited ...... 82

vi LIST OF TABLES

Table 1.1 List of courses samples in 2016 ...... 31

Table 1.2 List of courses samples in 2017 ...... 32

Table 1.3 Overwinter results and dates for each course ...... 33

Table 2.1 List of courses sampled for annual bluegrass composition and adult ABW in 2016 ....53

Table 2.2 List of courses sampled for annual bluegrass composition and adult ABW in 2017 ....54

Table 2.3 Annual bluegrass composition and adult ABW counts across all month and courses for 2016 ...... 55

Table 2.4 Annual bluegrass composition and adult ABW counts across all month and courses for 2017 ...... 59

Table 3.1 The arrangement of different turfgrasses in greenhouse for overwintered adult generation ...... 79

Table 3.2 The arrangement of different turfgrasses in greenhouse for spring adult generation ....80

Table 3.3 The arrangement of different turfgrasses in greenhouse for summer adult generation. 81

vii LIST OF FIGURES

Figure 1.1 Distribution map of sampled golf courses for 2016 and 2017 ...... 30

Figure 2.1 Adult ABW observed in plots across all months at different percentages of annual bluegrass for course 1 in 2016 (Cullasaja Club) ...... 44

Figure 2.2 Adult ABW observed in plots across all months at different percentages of annual bluegrass for course 1 in 2016 (Wildcat Cliffs) ...... 45

Figure 2.3 Adult ABW observed in plots across all months at different percentages of annual bluegrass for course 1 in 2016 (Highland Country Club) ...... 46

Figure 2.4 Adult ABW observed in plots across all months at different percentages of annual bluegrass for course 1 in 2016 (Burlingame) ...... 47

Figure 2.5 Adult ABW observed in plots across all months at different percentages of annual bluegrass for course 1 in 2016 (Cliffs at Walnut Cove) ...... 48

Figure 2.6 Adult ABW observed in plots across all months at different percentages of annual bluegrass for course 1 in 2017 (Cullasaja Club) ...... 49

Figure 2.7 Adult ABW observed in plots across all months at different percentages of annual bluegrass for course 1 in 2017 (Burlingame) ...... 50

Figure 2.8 Adult ABW observed in plots across all months at different percentages of annual bluegrass for course 1 in 2017 (Cliffs at Walnut Cove) ...... 51

Figure 2.9 Weed grid ...... 52

Figure 3.1 Grass arrangement in the greenhouse ...... 78

viii A REVIEW OF THE LITERATURE

The annual bluegrass weevil (ABW), Listronotus maculicollis Dietz (formerly Hyperodes sp. near anthracina, -anthracinus) is the single most destructive insect pest of golf courses in the northeastern United States and in certain provinces of Canada (Vittum et al. 1999; Simard et al.

2007). Damage from the larval stage of the weevil is observed on golf courses in short-moan cool season turf areas (Cameron and Johnson 1971). Damage is most severe on fairways, collars and tee boxes (McGraw and Koppenhoffer 2016). L. maculicollis was recently found in North

Carolina on golf courses with cool season turf in the western portion of the state. This weevil has been thoroughly studied since 1970 in the northeastern region of the United States; however, there is limited research on this weevil in the southeastern US. Therefore, the purpose of this work is to implement management approaches developed in the northeast and evaluate their efficacy on L. maculicollis in North Carolina as a golf course pest.

History, Origin, And Identification

The origin of this weevil pest in North America is currently unknown. Historical research suggests that ABW was described as a turf insect pest in Connecticut in 1931. The classification of ABW has undergone several genus and species revision since it was first discovered. This contributes to the difficulty in truly understanding the origin and history of this insect as a pest in North America.

The genus Listronotus is in the order Coleoptera, suborder Polyphaga, superfamily

Curculionoidea, family Curculionidae, and subfamily Cyclominae. Genus Listronotus has undergone many reclassifications since 1930. The reorganization of genus Hyperodes and

Listronotus has confusion among researchers and taxonomists (B.T Corbett personal observation). Kissinger (1964), states that the species within these two genera present complex

1 and challenging classification due to the similarities in both size and color. The two genera can be distinguished from each other by the second segment of the funiculus and the body length. In the genus Listronotus, the second segment of the funiculus is longer than the first segment, while the body is often more than 5 mm in length. In the genus Hyperodes, the second segment of the funiculus can be equal in size or longer than the first segment and the body of Hyperodes weevils is usually less than 4.5 mm in length according to Kissinger (1964). There is a total of 53 morphological characters that separates Listronotus and Hyperodes genera (Morrone 1997).

Currently, there are 32 species belonging to genus Listronotus in North America (O’Brien personal communication).

In 1957 and 1961 extension personnel at the Nassau County branch of the New York

State Cooperative Extension Service reported a weevil severely damaging and killing annual bluegrass (Poa annua L.) turf on golf courses (Cameron and Johnson 1971). Weevils found in the damaged areas were collected, then sent to the U.S National Museum (Washington D.C,

USA) and were identified by Dr. Rose Ella Warner as Listronotus anthracinus, formerly

Hyperodes anthracinus (Cameron and Johnson 1971; Warner 1965). From 1965 to 1967, golf course superintendents on Long Island, New York, and superintendents in Westchester County,

New York, reported damage from a similar weevil to turfgrass on several golf courses.

The origin of the pest in New York is still unclear; however, it is suspected that L. maculicollis have been on golf courses for several decades before the first report in 1965

(Cameron and Johnson 1971). Cameron and Johnson (1971) proposed that ABW have been on golf courses since they were first established in the Long Island and Westchester areas; however, damage was misidentified as other environmental factors. This thought for the origin of the

ABW in North America was based on reports of a Hyperodes weevil causing damage to grass on

2 golf courses in Farmington, Connecticut in 1931 (Britton 1932). The close proximity of both locations in New York City to the Connecticut locations indicates the weevil could be from the same locations since the 1930’s.

Britton (1932) sent weevil specimens from Farmington, Connecticut to the American

Museum of Natural History (Washington D.C, USA) for identification. A. H. Mutchler

(Taxonomist at American Museum of Natural History) identified the weevil as Hyperodes porcellus. Later, Stockton (1956) in his review of the genus Hyperodes noted that H. maculicollis had been collected on golf courses in Farmington, Connecticut. Stockton did not cite H. porcellus, indicating that the specimen collected by Britton in 1931 was later identified and changed to H. maculicollis at the American Museum of Natural History (Washington D.C,

USA).

In 1967, Henry Dietrich at Cornell University identified specimens from Long Island,

New York golf courses as L. maculicollis. Shread (1970) reported two Listronotus species causing damage to annual bluegrass in Connecticut as L. anthrosinous and L. maculicollis. L. anthrosinous had never appeared in previous taxonomic literature. Cameron and Johnson state that Shread suggested L. anthracinus as his finding. Shread reported both species were regularly found on golf courses at the same time. In 1967, L. anthracinus was the dominant species but in

1969 L. maculicollis was found more regularly in areas of damage. (Schread 1970). This lead to the first taxonomic study of these weevils conducted by Cameron and Johnson (1971).

Cameron and Johnson (1971) studies attempted to distinguish between the two species in their study in Connecticut and New York based on historical research on this genus; however, one of the species appeared to be a combination between L. maculicollis and L. anthracinus.

Stockton (1956) used male genitalia for positive identification for Listronotus species, however,

3 Dr. Warner stated there is little to no difference between L. anthracinus and L. maculicollis gentialia. Bissel (1937) and later Burke (1959) show that gentialia structures of Listronotus weevils (formally Hyperodes) are very similar to other weevils in different taxon. Warner

(1965) found the hairs and scales on the lateral regions of the prothoraces were the best characteristics for distinguishing species. Cameron and Johnson (1971) counted scales and hairs per unit area on the L. maculicollis and L. anthracinus specimens in the Cornell collection. Their study indicates that L. maculicollis had more hairs and fewer scales, while L. anthracinus had less hairs but more scales. The specimens from Long Island, NY were neither of the two species

(L. maculicollis, L. anthracinus) and later was classified as Listronotus sp near anthracinus based on the number of hairs and scales it obtained. Warner (1969) classified these specimens as

Litronotus sp near anthracinus.

The common name “annual bluegrass weevil” was first used by Shread (1970). Cameron and Johnson (1971) used the term “turfgrass weevil”. Vittum (1979) agreed with Cameron and

Johnson (1971), stating that “…since the two separate weevil species (L. maculicollis and L. anthracinus) feed on different turf species then the name turfgrass weevil is more accurate”. In

1980, the annual bluegrass weevil was still referred to as two separate species (Vittum 1980), however, in 1987 Vittum refers to the weevil as the “annual bluegrass weevil” (ABW) and only lists one species, Listronotus maculicollis (formerly Hyperodes maculicollis). In 1985, Dr. C.W.

O’Brien placed Hyperodes maculicollis in its current genus Listronotus, and designated the weevil as L. maculicollis (Vittum et al. 1999). Since 1987, observations of a Listronotous weevil feeding on cool season golf courses in the northeastern United States refers to the species as L. maculicollis and its common name is “annual bluegrass weevil”. Listronotus anthracinus has not reported causing damage in turfgrass since 1980.

4 Current Distribution

ABW are first reported to cause damage to cool season turfgrass in 1931 in Connecticut

(Cameron and Johnson 1971). Since then, it has expanded its range throughout the northeastern and mid-Atlantic United States, as well as some eastern Canadian provinces (McGraw 2016).

More recent studies have reported this pest presence in 14 states and two Canadian provinces

(McGraw and Koppenhofer 2016).

McGraw and Koppenhofer (2016) conducted the first distribution survey for ABW. They collected weevils from 293 courses (located in 14 states in the northeastern US and 2 Canadian provinces). These states include; Connecticut, Delaware, Maryland, Maine, New Hampshire,

New Jersey, New York, Ohio, Pennsylvania, Rhode Island, Massachusetts, Vermont, Virginia and West Virginia. There is evidence to support the concept that ABW populations are dispersing from the original site (New York City). Neither North Carolina nor Michigan were included in this distribution survey; however, ABW has been recovered from each of these states

(B.T Corbett personal observation).

General Biology and Seasonality

ABW go through two to three generations per year in the northeastern United States

(Vittum 1980; Vittum et al 1999). Metropolitan New York City is considered the possible origin of the pest (Vittum 1980). In more northern states (Connecticut, Vermont, and Maine) and some provinces of Canada north of New York City, ABW populations may only have one to two generations a year, while population in states south of New York City will typically complete three generations. The number of generations in the New York City area is typically determined by temperatures, rainfall and other environmental conditions (Vittum 1980; Vittum et al 1995).

5 ABW overwinter as adults in areas immediately adjacent to golf courses with overwintering sites including: mixed leaf litter, pine litter, tall grasses (rough), and natural or native areas (Cameron and Johnson 1971b, Vittum 1980, Vittum et al. 1999, Diaz 2006). Vittum

(1980) observed higher overwintering adult weevil populations in white pine litter; however,

Diaz (2006) found that mixed tree litter had the highest adult weevil populations followed by moss, high-cut grass, and pine litter. No overwintering adults were collected in preferred host plant areas (fairways, tees, and greens) in winter months (November-March); however, adult weevils were collected under trees adjacent to golf course fairways and in swards of grass

(Cameron and Johnson 1971b).

Adults are oblong, body length is usually double the body width, black and covered with fine hairs and yellow-brown and grayish-white scales. The scales on the elytra are in scattered groups, causing a faint mottled appearance (Cameron and Johnson 1971). As the adults grow older, many of the hairs and scales are worn off, leaving shiny black bodies. Occasionally, young and mature adults have been found with the distal quarter of each elytron an orange-brown color instead of the usual black (Cameron and Johnson 1971).

The timing of weevil emergence from overwintering areas often correlates with the time of year that forsythia (Forsythia spp.) and dogwood (Cornus spp.) begin to flower in the northeastern United States. This typically occurs from late March to early April. The flowering stage of the dogwood and forsythia reflects soil temperatures and air degree day accumulations that are a dependable indicator of adult migration from overwintering sites towards fairways

(Tashiro et al. 1978). Adults migrate from overwintering sites by walking instead of flying

(Rothwell 2003) even though they are capable of flight (Cameron and Johnson 1971; Schread

1970; Vittum 1980; et al. 1999). Adults can move considerable distances from overwintering

6 sites to the preferred feeding sites of short mown fairways (Diaz 2006). Adults travel up to 70 m from overwintering sites to the adjacent fairway on the golf course (Diaz and Peck 2007). The shorter mown turfgrass provides for higher rate of development, greater larvae fitness, and adult females prefer these areas for oviposition. (Rothwell 2003). Mowing of the turfgrass plant increases allocation of energy and nutrients to the leaves for new growth. This reallocation of resources may come from the nutrient and energy reserves located in the roots and crown of the plant making the crown and roots more favorable for ABW larvae survival (Reuss et al. 1983).

Adult females chew small notches in the turfgrass plant, typically on the sheaths of the plant .Females then deposit cylindrical eggs in the turfgrass sheath. Eggs are typically laid end to end, two to six eggs at a time. Egg are approximately three times longer than wide (.8 mm x .25 mm) and rounded at both ends appearing pale yellow to white with a transparent, colorless chorion. The chorion darkens to smoky black but it is still transparent revealing the embryo as it develops. ABW eggs typically hatch in four to five days (Cameron and Johnson 1971).

Larvae are crescent-shaped, wider in the center than at either ends, and have a dark- brown head capsule. They vary in length ranging from about 1 to 4.5 mm before they complete their development. The head capsule is distinct, with hypognathous mouth-parts. An inverted Y is located on the head of the larvae. The body is creamy white and the dorsal segment is composed of three fleshy lobes. The larvae undergo five instars, and the time of development varies depending on environmental conditions. During development, a single larva can destroy between 12 to 20 turfgrass stems by feeding near the crown of the plant (Cameron and Johnson

1971). Young larvae feed on the tissue within the protection of the stem. As larvae feed they outgrow the stem and later instar larvae drop to the soil surface where they chew on surface roots

7 and crowns. Larvae then pupate and emerge within seven to ten days as callow adults (Vittum

1980).

Male and female ABW are superficially identical, but upon closer observation, several differences can be noted. The third abdominal sternum of the females tends to bulge slightly in the middle, while the third sternum of the male is indented in the middle. The last sternum of the male is flattened and just slightly impressed at the apex, while the last sternum of the female has a large shallow depression. Although size does not distinguish the sex of an individual weevil, the average female ABW is slightly larger than the average male (Cameron and Johnson 1971;

Vittum 1980).

Host Plants

Vittum (1980) suggest that ABW feeding was restricted to annual bluegrass, P. annua. L.

Annual bluegrass is often considered a weed by many golf course superintendents. This grass weed is aggressive and can quickly invade and overtake areas of highly managed creeping bentgrass (Agrostis stolonifera) on golf courses. Annual bluegrass is primarily considered a

“winter annual”; however, due to constant low mowing heights, frequent irrigation, and significant fertilizer inputs applied on golf courses, this weed persists year-round through both seed and vegetative propagation (stoloniferous) (Michigan State University extension).

Superintendents spend significant time and money trying to eradicate annual bluegrass with little success (personal communication). The only way to eradicate this weed is to completely strip turf swards away and establish new sodded turf areas but can be time consuming and expensive. Many golf courses in the northeastern United States are becoming so contaminated with annual bluegrass that management strategies have evolved to effectively manage annual bluegrass as a playing surface rather than trying to eliminate it. One way to

8 accomplish this is with proper suppression of seed heads through plant growth regulators and appropriate management strategies. (Michigan State University extension).

ABW have often reported damage to annual bluegrass while other turfgrass species nearby (creeping bentgrass, Agrostis spp; Kentucky bluegrass, Poa pratensis L.) show few signs of damage (Cameron and Johnson 1971a, b, Vittum 1980, and Vittum et al. 1999). Rothwell

(2003) observed that numbers of adult and larval found in field plots of annual bluegrass and creeping bentgrass were similar, indicating that weevils are recovered from both species of turfgrass. Rothwell (2003) and Kostromytska (2014) established that adult ABW preferred annual bluegrass over creeping bentgrass for oviposition. Larvae that fed on annual bluegrass weighed more than larvae feeding on creeping bentgrass (Rothwell 2003; Kostromytska 2014).

McGraw and Koppenhofer (2009) found that unmixed stands of creeping bentgrass were more tolerant to weevil damage than mixed stands of annual bluegrass and creeping bentgrass. On established golf course fairways, there was no effect of host species density (annual bluegrass vs creeping bentgrass) on adults and larval density (McGraw and Koppenhofer 2009).

9 Management

CULTURAL CONTROL

ABW initially appeared to cause damage to annual bluegrass while other turfgrass species nearby (creeping bentgrass and Kentucky bluegrass) showed no signs of damage

(Cameron and Johnson 1971, Vittum 1980, and Vittum et al. 1999). By contrast, a more recent study showed that the number of adult and larval ABW recovered from field plots of annual bluegrass and creeping bentgrass were similar, indicating that ABW can be found in both species of turfgrass (Rothwell 2003). Additional studies indicate that ABW prefer annual bluegrass for oviposition, when compared to creeping bentgrass (Rothwell 2003 and Kostromytska 2014).

Adult ABWs feeding on annual bluegrass weighed more and females laid more eggs than ABW feeding on creeping bentgrass (Rothwell 2003 and Kostromytska 2014). Unmixed stands of creeping bentgrass are more tolerant to weevil damage than mixed stands of annual bluegrass and creeping bentgrass (McGraw and Koppenhofer 2009). In established golf course fairways, there was no effect of host species (annual bluegrass or creeping bentgrass) on adults or larval density (McGraw and Koppenhofer 2009).

Rothwell (2003) compared different varieties of bentgrass and Kentucky bluegrass to annual bluegrass for ABW survivability and host preference. Kostromytska et al (2014) compared ABW survivability and host preference on bentgrass varieties most commonly found on old and new golf courses. The results found that ABW laid more eggs in annual bluegrass than Kentucky bluegrass and different bentgrass varieties. Additionally, there were significantly more feeding scars and ovipositional probes on annual bluegrass compared to other trugrasses.

Overwintered females laid 19 times more eggs while summer-generation females laid five times more eggs in annual bluegrass, compared with the preferred bentgrass cultivar. These results

10 suggest that cultural control for ABW could be achieved by planting non-susceptible host species on golf courses.

Cultural control of ABW can be significantly suppressed by adjusting the mowing height of the turfgrass (Rothwell 2003). The majority (69%) of the damage on golf courses is most commonly found in fairways and less than 10% of damage is seen in roughs (McGraw and

Koppenhofer 2016). ABW damage is associated with turfgrasses maintained below 1.25 cm in height (Rothwell 2003, Kostromytska and Koppenhofer 2014). Turfgrasses above this cut height had little damage, with a lower survivability of ABW. Though this technique of cultural control has positive results, golf courses are trending the opposite way as mowing heights are lowering with advancements in mowing equipment and clientele preferences.

NATURAL AND BIOLOGICAL

The only natural enemies affecting immature ABW are nematodes (Vittum 1980; Grant and Rossi 2004; McGraw and Koppenhöfer 2007, 2008a). McGraw (2009) found two species of nematode, Steinernema carpocapsae Weiser and Heterorhabditis bacteriophora Poinar infecting weevil stages from third instar to adults. Both nematode species exhibited host response, appearing in high densities the weeks immediately following high densities of first generation

ABW larvae in the soil. However, both species failed to reduce ABW populations in field trials.

Other research indicated the nematode Steinernema carpocapsae can reduce populations of larvae by 50% (Vittum 1999).

Few statistically significant relationships supported McGraw’s (2009) hypothesis that weevil spatial dispersion influences entomopathogenic nematode spatial dispersion. Nematode seasonal occurrence and generational impact variability and their lack of spatial association with

11 ABW suggest an inability of nematode populations to reliably control weevil populations on golf course fairways (McGraw 2009).

Vittum and McNeill (1999) examined the potential of Microctonus aethiopoides Loan

(parasitic wasp of the alfalfa weevil) as a biological control agent, but wasp parasitism only resulted in 20%mortality of ABW larvae under laboratory conditions.

CHEMICAL CONTROL

Targeting adults as they emerge from overwintering sites in the early spring is the most effective management strategy using synthetic insecticides for ABW (Vittum et al 1999). Adults emerge from overwintering sites adjacent to the golf course fairway and migrate onto the course fairways, tees and greens. Adults chew small holes or notches in the turfgrass leaf blade.

Females deposit fertilized eggs in these notches that later hatch into larvae. Larvae feed and tunnel further down the turfgrass stem until they reach the crown and roots of the plant in the soil. Adult feeding is relatively insignificant and does not affect the turfgrass plant. The larval stage causes the most serious damage from ABW (Cameron and Johnson 1971).

Laboratory and field-plot tests were first conducted in 1968 and 1969 to find a chemical control for the annual bluegrass weevil (Cameron 1970). Golf courses in northeastern US apply an average of 3.9 applications per year to manage adults and larvae (McGraw 2016). Recent studies show that 65-79% of surveyed superintendents currently use pyrethroids and chlorpyrifos

(Dursban) to treat ABW adults (McGraw 2016). In 2005, previous to pyrethroid resistance, pyrethroids provided over 85% control of adults (McDonald 2005). Pyrethroids, particularly bifenthrin, (Talstar), have been used for over 40 years on golf courses for the control of the

ABW in the northeastern US. This resistance to bifenthrin has also been observed in western

North Carolina after five years at three applications a year for ABW management (Dr. Billeisen

12 unpublished data). Currently, chlorantraniliprole (Acelepryn) and cyantraniliprole (Ference) are used to target ABW larvae due to their length of residual and ability to control multiple white grub species. Effective alternative larvicides to ABW include indoxacarb, trichlorfon, spinosad, neonicotinoids, and combination products (e.g., bifinthrin + imidacloprid) (McGraw 2016).

Preventative ABW control in sites known to have chronic infestations may include long residual insecticides before egg hatch such as imidacloprid (Merit) (McDonald 2005).

In northeastern United States, there are typically 2-3 generations per year, so targeting each generation is key to successfully suppressing the ABW. Overwintered adults emerge when forsythia is in full bloom. Preventative adulticides (pyrethorids, chlorpyifos) are typically applied when adult ABW are seen walking across turf areas in the spring (McDonald 2005). These are applied to the perimeter of the fairways, green and tees. Overwintered adults not controlled by initial spray mate and start to lay eggs around the time the eastern redbud and flowering dogwood start to bloom. Once the dogwood is in full bloom is when a preventative larvicide

(Acelepryn,erence) is needed. Larvicides (indoxacarb, trichlorfon, spinosad) can be used in late

May once the Rhododendron (R. catawbiense) has fully bloomed to control any additional larvae.

The next generation adults emerge in mid to late June. Another adulticide, Provaunt (Conserve) is typically needed to target adults. The larvae from this new generation appear in late July into early August. Another larvicide is typically needed although it is recommended to apply an alternative larvicide than the one previously applied earlier in the spring. One last adulticide is needed at the end of the August into early September to target any remaining adults before they return to the overwintering sites. This process of monitoring and rotating chemistry is the best current management approach to reduce pest pressure with resistance.

13 THE PURPOSE AND FOCUS OF THIS RESEARCH

ABW were first observed feeding on cool season turfgrass at golf courses in western

North Carolina in 2006 (B.T Corbett personal communication). Limited data on ABW biology and ecology is available for this region and reliance upon numerous studies from northern climates is not dependable. Very little information was available concerning ABW distribution, overwintering sites, the number of generations per year and cost-effective management programs in the southeastern United States. The following objectives were selected to govern the development of a research program studying the ABW distribution in the mountains of North

Carolina. The first objective was to look at the distribution of ABW in western North Carolina where cool season grasses are the dominant turf species and to determine if ABW overwintering habitats are similar to ones found in previous research in the northeastern U.S. The second objective was to determine the impact of different turfgrass species quantities on adult ABW prevalence on golf courses. The final objective was to look at grass tolerance (damage to grass), antibiosis (larval survival and growth) and antixenosis (resistance to feeding) on three different turfgrass cultivars commonly found in western North Carolina. Data obtained from studies associated with these three objectives would provide guidance in the development of control strategies for ABW on golf course techniques in western North Carolina. In addition, a collaborative project with the Smithsonian Institute was developed to classify the species present in North Carolina as compared to those from areas in the northeastern United States.

14 Literature Cited

Britton, W.E. 1932. Weevil grubs injure lawns, Bull. Conn. Agric. Exp. Station, 338:593.

Cameron, R. S., and N. E. Johnson. 1971. Chemical control of the “annual bluegrass weevil,” Hyperodes sp. nr. anthracinus. J. of Econ. Entomol. 64(3): 689-693.

Cameron, R.S., and N. E. Johnson. 1971b. Biology of a species of Hyperodes (Coleoptera: Curculionidae): a pest of turfgrass. Search Agriculture 1: 1–31

Cowles, R.S., A.M. Koppenhöfer, B.A. McGraw, S.R. Alm, D. Ramoutar, D.C. Peck, P. Vittum, P. Heller, and S. Swier. 2008. Insights into managing the annual bluegrass weevils. Golf Course Manage. August: 86−92.

Diaz, M.D.C. 2006. Population dynamics, phenology and overwintering behavior of the annual bluegrass weevil, Listronotus maculicollis Dietz (Coleoptera: Curculionidae), in the Golf Course Landscape. MS thesis. Cornell University, Ithaca, NY.

Diaz, M.D.C., and D.C. Peck. 2007. Overwintering of annual bluegrass weevils, Listronotus maculicollis, in the golf course landscape. Entomol. Exp. Appl. 125: 259−268.

Doskocil, J. 2009. Annual Bluegrass Weevil. NCSU Cooperative Extension. https://www.ces.ncsu.edu/depts/ent/notes/O&T/lawn/note146/note146.html

Grant, J.A., and F.S. Rossi. 2004. Evaluation of reduced chemical management systems for putting green turf, pp. 1-13. In J.L. Nus [ed.], 2004 Turfgrass and Environmental Research Summary. USGA Green Section, Far Hills, NJ.

Kissinger, D.G. 1964. Curculionidae of America North of Mexico. A key to the Genera. Taxonomic Publications, South Lancaster. 143 pp.

Kostromytska, O.S. and Koppenhöfer, A.M. 2014 Ovipositional preferences and larval survival of annual bluegrass weevil, Listronotus maculicollis, on Poa annua and selected bentgrasses (Agrostis spp.). Ent. Exp. et App 152, 108–119.

15 McDonald, S. J., and P. H. Dernoden. 2005. Biology and management of the annual bluegrass weevil. University of Maryland Turfgrass Technical Update TT-52.

McGraw, B. A. 2009. The Ecology and the Biological Control of the Annual Bluegrass Weevil, Listronotus maculicollis Kirby (Coleoptera: Curculionidae) Using Entomopathogenic Nematodes (Rhabditida: Steinernematidae and Heterorhabditidae). Ph. D. dissertation, Rutgers, The State University of New Jersey, New Brunswick.

McGraw, B. A., and A. M. Koppenhöfer. 2016. A Survey of Regional Trends in Annual Bluegrass Weevil (Coleoptera: Curculionidae) Management on Golf Courses in Eastern North America. J. of Int. Pest Man, 8(1).

(MSU Extension) Ronald Calhoun, Michigan State University Department of Crop and Soil Science. Turf Weeds, Annual Bluegrass weed Id guide.

Morrone, J.J. 1997. Cladistics of the New World genera of Listroderina (Coleoptera: Curculionidae: Rhytirrhinini). Cladistics. 13: 247-266.

Ruess RW, McNaughton SJ, Coughenour MB 1983. The Effects of Clipping, Nitrogen Source and Nitrogen Concentration on the Growth Responses and Nitrogen Uptake of an East African Sedge. Oecologia 59:253–261

Rothwell, N. L. 2003. Investigation into Listronotus maculicollis (Coleoptera: Curculionidae), a pest of highly maintained turfgrass. Ph.D. dissertation, University of Massachusetts, Amherst (Mass.).

Schread, J.C. 1970. The annual bluegrass weevil. Conn. Agric. Exp. Sta. Circ. no. 238, New Haven, CT.

Simard, L., J. Brodeur, and J. Dionne. 2007. Distribution, abundance, and seasonal ecology of Listronotus maculicollis (Coleoptera: Curculionidae) on golf courses in Quebec, Canada. J. Econ. Entomol. 100: 1344−1352.

Stockton, W.D. 1956. A review of the Nearctic species of the genus Hyperodes Jekel (Coleoptera: Curculionidae). Ph.D. Thesis, Cornell University, Ithaca, NY. 122pp.

16 Tashiro, H., C.L. Murdoch, R.W. Straub, and P.J. Vittum. 1978. Evaluation of insecticides on Hyperodes sp., a pest of annual bluegrass turf. J. Econ. Entomol. 70: 729−733.

Vittum, P.J. 1980. The biology and ecology of the annual bluegrass weevil, Hyperodes sp. near anthracinus (Dietz) (Coleoptera: Curculionidae). Ph.D. thesis. Cornell Univ., Ithaca, NY. 164

Vittum, P.J. and H. Tashiro. 1987. Seasonal activity of Listronotus maculicollis (Coleoptera: Curculionidae) on annual bluegrass. J. Econ. Entomol. 80:773-778.

Vittum, P. J., M. G. Villani, and H. Tashiro. 1999. Turfgrass insects of the United States and Canada. Cornell University Press, Ithaca, NY.

Vittum, P.J. and M.R. McNeill. 1999. Suitability of Listronotus maculicollis (Coleoptera: Curculionidae) as a host for Microctonus hyperodae (Hymenoptera: Braconidea). J. Econ. Entomol. 92(6):1292-1300.

Warner, R.E. 1965. Hyperodes anthracinus (Dietz) damaging golf greens (Coleoptera: Curculionidae). Coleopts. Bull. 19: 32.

17 CHAPTER 1

Investigation of Listronotus maculicollis distribution in Western North Carolina.

The annual bluegrass weevil (ABW), Listronotus maculicollis Kirby (Coleoptera:

Curculionidae) is a major insect pest of highly maintained turfgrass in the northeastern United

States and in some provinces of Canada. In recent years, this destructive weevil has been found in western North Carolina where cool-season grasses are the dominant turf species on golf courses. To determine the distribution of this pest in western North Carolina, 46 golf courses were sampled in 2016 and 2017. Sampling was conducted using a one square meter area soapy water disclosing solution directly applied to the turfgrass on fairways, tees and greens. Sampling was conducted from May to August in 2016 and May to September in 2017. Adult annual bluegrass weevils were detected on 32 golf courses.

18 Introduction

The annual bluegrass weevil (ABW), Listronotus maculicollis Kirby (formerly

Hyperodes sp. near anthracina, -anthracinus) is a pest of close-cut turfgrasses found on golf courses in northeastern United States (Vittum et al. 1999), and some areas of Ontario and

Quebec, Canada (Simard et al. 2007). ABW was first reported to damage cool season turfgrass in

1931 in Connecticut (Cameron and Johnson 1971). Since then, it has expanded its range throughout the Northeastern and Mid-Atlantic United States, as well as some eastern Canadian provinces (McGraw and Koppenhofer 2016). More recent studies have reported this insect pest in 14 states and two Canadian provinces (McGraw and Koppenhofer 2016).

ABW larvae can cause severe turf damage on tees, fairways, collars and greens that contain annual bluegrass (Cameron and Johnson 1971; Vittum 1980, McGraw 2009; McGraw and Koppenhofer 2016). Annual bluegrass (Poa annua L.) is preferred by egg-laying females and is susceptible to larval feeding (Rothwell 2003; Kostromystka and Koppenhofer 2014).

Adult females oviposit eggs in the turfgrass sheath with usually two to six eggs laid at a time

(Cameron and Johnson 1971). Young larvae feed on the tissue within the protection of the stem.

As larvae feed they outgrow the stem and the later instar descend into the soil surface where they feed on surface roots and crowns. This feeding is detrimental to the turfgrass plant, causing yellowing and eventually death. A single larva can damage between 12 to 20 turfgrass stems

(Cameron and Johnson 1971). Significant damage develops when population of ABW exceed

100 larvae/0.1m2 (McGraw and Koppenhofer 2016).

ABW overwinter as adults in areas adjacent to the fairways on the golf course.

Overwintering sites include: mixed leaf litter, pine litter, tall grasses (rough) and natural or native areas (Cameron and Johnson 1971b, Vittum 1980, Vittum et al. 1999, Diaz 2006). Overwintered

19 adults emerge in the spring, typically from late March to early April in western North Carolina.

Adults migrate away from overwintering areas into short mown turfgrass areas. Damage from larvae is typically observed from late May until early September (Vittum 1999).

Damage from larvae is observed to be most common on collars of greens and on the putting surface (Cameron and Johnson 1971); however, recent research suggests the most severe damage occurs on the edge of fairways (McGraw and Koppenhofer 2016). Damage is not generally seen on putting greens due to low mowing heights, mowing frequency and the frequency of pesticides used in these areas (McGraw and Koppenhofer 2016).

McGraw and Koppenhofer (2016) conducted the first distribution survey for ABW. They surveyed 293 golf courses located in 14 states in the US and two Canadian provinces. The survey included: Connecticut, Delaware, Maryland, Maine, Massachusetts, New Hampshire, New

Jersey, New York, Ohio, Pennsylvania, Rhode Island, Vermont, Virginia and West Virginia.

There is evidence to support the concept that the ABW populations are dispersing over time in all directions away from the original site (New York City).

This distribution survey is helpful to better understand the distribution of the pest in the northeast U.S.; however, there is limited information on the biology, ecology and distribution of this pest in North Carolina which was not included in that study. ABW was first observed in western North Carolina in 2006 (Allen Hurns, personal communication), yet the exact distribution of the pest in North Carolina is unknown. The main objective of this study is to do an extensive survey of western North Carolina to better understand the geographic distribution of

ABW. A second objective is observing overwintering sites in western North Carolina and comparing the location and composition of these sites to information from studies in northeastern

United States. The data obtained from this study would provide insight to the severity and

20 distribution of the pest in western North Carolina, and document information on the overwintering preference of adult ABW.

Materials and Methods

SURVEY

In the spring of 2016, 25 golf course superintendents in western North Carolina were contacted and arrangements made to sample for ABW on their respective courses and in 2017,

21 additional superintendents were contacted. The purpose of the collaboration was to establish a sampling survey across golf courses in western North Carolina.

ABW adults were samples in three distinct locations in the mountain of North Carolina.

The mountains were divided into three regions, central, southern and northern (figure 1.4). The first-generation adults can be seen on golf courses in mid to late June.

In 2016, sampling in the southern region was conducted on 23 May-5 June, sampling in the central region was conducted on 6-15 June, and sampling in the northern region was conducted on 20-24 June. Golf courses where ABW presence was confirmed were not revisited in 2016. Additionally, golf courses with no observed ABW presence were revisited in July and

August of 2016 and resampled.

In 2017, 21 different golf courses were sampled along with 10 golf courses where no

ABW were found in 2016. Sampling in 2017 started in mid-May due to an unseasonably warm winter and spring. The southern region was sampled on 15-25 May. The central region was sampled on 30 May-9 June, followed by the northern region, which was sampled on 12-23 June.

Golf courses without ABW were revisited in July and August to confirm that ABW adults were not missed in previous sampling.

21 Four fairways were sampled on the initial visit to each golf course. The sampling on each golf fairway was initiated at the tee box then moving along the fairway towards the putting green and concluded at the putting green. ABW adults were sampled using a soapy water flush that was conducted by adding Joy lemon-scented dish liquid (14.7 mL) in water (3.7L) in a garden watering can to apply the liquid solution directly to a 1 m2 area. Samples were taken 30-45 m apart in a line along each fairway. The number of samples varied for each fairway due to the length of the fairway from the tee to the putting green; however; a minimum of nine samples were taken on each golf fairway. An average of 36 samples were taken at each golf course. After applying the soap solution to the turfgrass area, the grass surface was examined closely for ABW presence for three minutes before moving to the next sampled area. All observed ABW were hand collected from each turfgrass plot, recorded and then placed in vials containing 95% ethyl alcohol and returned to the laboratory. ABW adults found were submitted to the North Carolina

State University museum for taxonomic comparison to look for variations in morphology and genetic base pairs from each course. If no ABW were found in initial sampling, the course was revisited the following month and four different fairways were chosen for sampling at that golf course. Fairways were chosen based on locations where there was habitat favorable to ABW based on previous research conducted in the northeastern U.S. These characteristics included such as slope, large quanities of annual bluegrass, overwintering sites and history of damage.

Sampling at these sites had a greater likelihood for finding ABW on the golf courses. All sampling was conducted on the perimeter of fairways. The fairway perimeter is generally where the shorter-mown turfgrass (<1.27 cm mowing height) meets the higher, thicker turfgrass known as the rough (> 3.175 cm mowing height). The fairway perimeter generally contains the largest

22 concentrations of annual bluegrass and is where overwintered adults first travel to start feeding and mating in the spring. This increased the probability of finding adult ABW.

OVERWINTERING SITES

Three golf courses Burlingame (Transylvania County)(35.11539, -82.98029), Cullasaja

Club(Macon County)( 35.08616, -83.53687), Etowah Golf Club(Henderson County)( 35.32319,

-82.6054) where ABW were found in 2016 mapping survey were revisited in late 2016 and early

2017 for sampling of potential overwintering sites for ABW. The sampling was conducted in

November and December (2016), and January-March (2017). There were a total of four different types of overwintering materials that were examined. These included 1.) leaf litter from white pine trees (Pinus strobus L.), 2.) eastern hemlock (Tsuga canadensis L.) pine litter Carr.),3.) mixed leaf litter and 4.) native grasses.

Samples of different pine litter and native grasses (930 cm2) were collected at least once a month from beneath selected tree species and in native grass areas. The litter was collected using a steel tine leaf rake. Litter and native grass areas were raked into piles and collected by hand.

Samples were placed in brown paper bags and transported to the Lake Wheeler Turf Research

Field Lab (Raleigh, NC, USA). Each sample was placed in a 110 L portable ice chest. Ice chests were lined with paper toweling, covering the bottom and sides. All litter and native grass was removed from brown paper bags and placed in the ice chests. Ice chests were filled with lukewarm water (36-40 degrees C), submerging the material. Paper toweling was placed on water surface and the cooler lid was closed. Adults and paper toweling were removed and new paper toweling was applied every 30 minutes to collect any emerging adults from the material.

Each sample was left submerged for one and a half hours before removing material. This method was modified from Vittum (1980).

23 Results and Discussion

MAPPING SURVEY AND OVERWINTERING

ABW adults were detected on 31 out of 46 golf courses across the three regions. Adult

ABW were found in all three sampling regions. Twenty-one courses were sampled in the southern region and ABW were found on nineteen of those courses. Weevils were detected on six of the eleven courses sampled in the central region. ABW were found on six of the twelve courses sampled in the northern region. Sixty-seven percent of the golf courses sampled in this survey were infested with ABW. The highest percent of golf courses infested with ABW were observed in the southern region with 90% of the golf course having ABW. ABW were found on

55% of the golf courses in the central region and 50% of the golf courses sampled in the northern region. These data show ABW is a widespread pest in western North Carolina and is not isolated to any specific area.

ABW have been found in over 14 states and yet they primarily move by walking and are not capable of flight (McGraw and Koppenhofer 2016, Vittum 1980). The expansion of their territory have just recently been discovered with the bulk of research being conducted in the last

15 years. With this weevil first observed in North Carolina in 2006, ABW could have been here years before that date. It is possible that ABW was introduced into these new states via sod. In

North Carolina 15 out of 31 golf courses sampled that imported sod from the northeast had

ABW. North Carolina also provides a suitable habitat for ABW survival. In western North

Carolina temperatures provide the proper conditions for cool season grasses and plenty of rainfall. The southern region had the highest percentage of golf courses where ABW were found.

The climate in this region receives bountiful rainfall and is classified as a temperature rainforest.

These conditions make for a favorable environment for ABW and their host plants. Both

24 creeping bentgrass and annual bluegrass thrive in areas with adequate temperature and moisture.

The discovery of this pest in western North Carolina is not a surprise especially in the southern region. With many neighboring states reporting ABW it was a matter of time before it was seen here.

ABW pyrethroid resistance populations have been found in the northeast since 2008

(Cowles 2008). Insecticides have changed over the last 40 years. Historically, golf course superintendents have used organophosphates and carbamates for broad spectrum control of many golf course insect pests. In the past 20 years, golf course superintendents have switched to more narrow spectrum chemical approaches due to EPA restrictions and public health concerns

(Vittum GCM 2008). These insecticides are much safer but more expensive. Pyrethroids; however, have remained economical and provide control for many golf course insect pests in western North Carolina: these include black cutworms (Agrotis ipsilon), fall armyworm

(Spodoptera frugiperda), white grubs, black turfgrass ataeniu (Ataenius spretulus) and nuisance ants (Lasius neoniger). These products have been the sole reliance for many golf course superintendents because of their great control and affordability. It is no coincidence that the discovery of ABW in many states occurred from 2005 to 2010, the same time pyrethroid resistance to this insect was first observed. The frequent spraying of pyrethroids has caused resistance to ABW. It could also be the reason ABW was not a pest in many states until now. In

North Carolina the discovery of resistance population of ABW have already been found

(Billenisn unpublished data). This theory explains why ABW were suppressed for so long but in the last 5 to 10 years are finally being seen in many new areas.

White pine litter is the preferred overwintering sites in the New York City area for ABW

(Vittum 1980). This could be because this tree species is commonly found on many of the golf

25 courses in the northeastern United States. However, research conducted by Diaz (2006), suggests that mixed leaf litter provides the best habitat for overwintering sites. This project collected samples from different potential overwintering sites for ABW and the samples were then processed and observed for the presence of adults. In North Carolina, more adult ABW were found in white pine straw for an overwintering site over other choices.

A total of 12 samples were taken at each course (three samples from each of the four overwintering sites). Samples collected under white pine trees across the three courses and all sampling dates had a total of 61 adults recovered (5.08 per sample), eastern hemlock samples contained 34 adults (2.83 per sample), native grass samples contained 14 adults (1.16 per samples) and samples of mixed leaf litter contained 12 adults (1 per sample). Adults were recovered on paper toweling or on the sides of the coolers. A total of 121 adult weevils were recovered during four out of five months (November, December, January, and February), with no adults being recovered in the month of March. Out of the 121 adults recovered, 86% were recovered within the first 30 minutes after leaf litter was submerged. All the adults that were recovered were found within the first hour of the leaf litter being submerged with no weevils being recovered after an hour.

26 Literature Cited

Cameron, R. S., and N. E. Johnson. 1971. Chemical control of the “annual bluegrass weevil,” Hyperodes sp. nr. anthracinus. J. of Econ. Entomol. 64(3): 689-693.

Cameron, R.S., and N. E. Johnson. 1971b. Biology of a species of Hyperodes (Coleoptera: Curculionidae): a pest of turfgrass. Search Agriculture 1: 1–31

Maier, R.R, and Potter D.A. 2005. Factors Affecting Distribution of the Mound-Building Ant Lasius neoniger (Hymenoptera: Formicidae) and Implications for Management on Golf Course Putting Greens. J. Econ. Entomol. 98(3): 891-898.

Rothwell, N. L. 2003. Investigation into Listronotus maculicollis (Coleoptera: Curculionidae), a pest of highly maintained turfgrass. Ph.D. dissertation, University of Massachusetts, Amherst.

Silcox, C.A. and Vittum P.J. 2008. Turf Insecticide modes of action and resistance management. Golf Course Management Magazine Sept. 82-90.

27 Vittum, P.J. 1980. The biology and ecology of the annual bluegrass weevil, Hyperodes sp. near anthracinus (Dietz) (Coleoptera: Curculionidae). Ph.D. thesis. Cornell Univ., Ithaca, NY. 164.

Vittum, P. J., M. G. Villani, and H. Tashiro. 1999. Turfgrass insects of the United States and Canada. Cornell University Press. Ithaca, NY.

28 Figure 1.1 Map of ABW distribution in western North Carolina on golf courses sampled in

2016 and 2017

29 Table 1.1. List of golf courses sampled in 2016

ABW Adults 2016 Latitude Longitude Region Presence Found Highland Falls Country Club 35.07076 -83.18478 South Y 32 Etowah Valley Golf Club 35.32319 -82.60541 South Y 27 Linville Ridge 36.10133 -81.85692 North Y 12 Mountaintop Golf and Lake Club 35.12301 -83.138 South Y 9 Burlingame Country Club 35.11539 -82.98029 South Y 63 Linville Land and Harbor Golf Club 36.04253 -81.90181 North N N/A Roaring Gap 36.40293 -80.98596 North N N/A Mountain Glen Golf Club 36.12821 -81.93762 North N N/A Cliffs at Walnut Cove 35.46269 -82.60232 Central Y 44 Biltmore Forest Country Club 35.53207 -82.53687 Central N N/A Cullasaja Club 35.08616 -83.16467 South Y 21 Hendersonville Country Club 35.3054 -82.48584 South Y 39 Diamond Creek 36.15528 -81.83773 North Y 16 Grandfather Golf and Country Club 36.09662 -81.85579 North N N/A Omni Grove Park Inn 35.61986 -82.54662 Central Y 8 Laurel Ridge Country Club 35.48688 -83.01313 Central N N/A Sequoyah National Golf Club 35.43184 -83.32893 Central Y 5 Maggie Valley Club and Resort 35.52268 -83.04814 Central Y 30 Linville Golf Club 36.06555 -81.86811 North N N/A Wildcat Cliffs Country Club 35.0861 -83.15286 South Y 48 Highlands Country Club 35.05284 -83.21732 South Y 13 Country Club of Asheville 35.64056 -82.55505 Central N N/A Olf Edwards Club 35.1047 -83.16123 South Y 15 Black Mountain Golf Club 35.62626 -82.33379 Central N N/A Wolf Laurel Country Club 35.96844 -82.50119 North N N/A

30 Table 1.2. List of golf courses sampled in 2017

Longitud ABW Adults 2017 Latitude Region e Presence Found Wade Hampton Golf Club 35.0865 -83.07058 South Y 9 Headwaters Golf Course 35.08572 -83.0728 South Y 23 Beech Mountain 36.21095 -81.889 North Y 17 Asheville Municipal Golf Club 35.57837 -82.50088 Central N N/A Bear Lake Reserve Golf Club 35.22113 -83.05794 South Y 5 High Hampton Inn and Country Club 35.09922 -83.08411 South Y 31 The Ridges Golf Club 35.04349 -83.73224 South Y 6 Mountain Air Country Club 35.87068 -82.34645 North Y 2 Elk River Club 36.1568 -81.89826 North Y 83 High Meadows Country Club 36.40514 -80.99781 North N N/A Boone Golf Club 36.19258 -81.64739 North N N/A Champion Hills Club 35.29334 -82.51596 South Y 56 Kenmure Country Club 35.25467 -82.44472 South Y 5 Balsam Mountain Preserve 35.39837 -83.12246 South Y 22 Trillium Links and Lake Club 35.14401 -83.13953 South Y 14 Cummings Cove Golf & Country Club 35.30856 -82.56608 South Y 3 Sapphire Valley Resort 35.12505 -83.05758 South Y 7 Sapphire National Golf Club 35.11324 -83.02517 South Y 28 Lake Taxaway Country Club 35.13788 -82.9547 South Y 25 Connestee Falls Golf Club 35.14331 -82.72705 South Y 7 Blowing Rock Country Club 36.12195 -81.6639 North N N/A

31 Table 1.3. Overwintering Sampling

Overwintering Sites Number of Adults Found Total

11/17/16 12/6/16 1/26/17 2/16/17 3/14/17

Etowah Country Club

Mixed Leaf Litter 1 2 0 0 0 3 White Pine Straw 4 11 6 2 0 23 Eastern Hemlock Pine Straw 1 1 7 1 0 10 Native grasses 0 3 0 0 0 3

Burlingame Country Club

Mixed Leaf Litter 0 0 0 0 0 0 White Pine Straw 3 4 2 6 0 15 Eastern Hemlock Pine Straw 1 0 2 1 0 4 Native grasses 0 0 0 1 0 1

Cullasaja Club

Mixed Leaf Litter 3 0 2 0 0 5 White Pine Straw 9 11 8 5 0 33 Eastern Hemlock Pine Straw 2 6 10 2 0 20 Native grasses 1 3 5 0 0 9

32 CHAPTER 2

Presence of ABW Associated With Host Plant Abundance

The annual bluegrass weevil (ABW), Listronotus maculicollis Kirby, is a native pest of highly maintained turfgrass in the northeastern United States and specific provinces of Canada, since 1931 there have been reports of ABW damage of turfgrass on golf courses in North

America. Previous research and observations indicate ABW are more abundant in annual bluegrass (Poa annua L.) than other turfgrass species including creeping bentgrass (Agrostis stolonifera). In 2006, ABW presence and damage to annual bluegrass on golf courses was reported in western North Carolina. In 2016 and 2017, golf courses in western North Carolina having reported ABW damage were sampled for adult distribution in mixed stands of creeping bentgrass and annual bluegrass. The sampling was conducted by using a soap water flush within a 1 m2 grid. The turfgrass species composition was determined using a grid containing 225 6 cm2 squares (figure 2.9). Data collected from this study in the summers of 2016 and 2017 indicate there is not significant evidence to support adult ABW populations will be found in areas containing higher percentages of annual bluegrass. This study indicates that adults are widely distributed on golf course fairways independent of turfgrass species.

33 Introduction

ABW is a pest of highly-maintained turfgrass in the northeastern United States and

Canada (Vittum 1999). Most of the damage to turfgrass is caused by the larval stage of this insect pest. Larvae go through five instars before pupating. Young larvae (1st-3rd instar) feed on the tissue inside the stem while later instars (4-5 instar) descend from the plant into the soil and feed on the roots. The fifth instar causes the most severe damage of turfgrass on golf courses

(Tashiro and Straub 1973). During development, a single larva can destroy between 12 to 20 turfgrass stems (Cameron and John 1971). Adults create notches in the turfgrass blades, weakening the grass; however, since this feeding is above the meristem, adult feeding causes no severe damage (Cameron and Johnson 1971).

Damage from ABW is only observed in highly-maintained, close-cut turf (Vittum et al

1999). Previous research suggests that ABW cause more damage to annual bluegrass on golf courses than to adjacent creeping bentgrass (Agrostis stolonifera L.) (Cameron 1970, Cameron and Johnson 1971, Vittum 1980, Vittum et al 1999). Vittum (1980) determined that ABW fed only on annual bluegrass turf species on golf courses; however, most of the feeding studies were conducted in the laboratory. Most weevil damage is within swards of annual bluegrass while immediately adjacent swards of creeping bentgrass are untouched. (Rothwell 2003).

ABW can be found feeding in both creeping bentgrass and annual bluegrass. Rothwell

(2003) observed that adult and larval numbers found in field plots of annual bluegrass and creeping bentgrass were similar even though damage is not observed in creeping bentgrass.

Research from Rothwell (2003) and Kostromytska (2014) suggest adults prefer annual bluegrass turf over creeping bentgrass turf for oviposition and larval feeding. Larvae that fed on annual bluegrass turf outweighed larvae feeding on creeping bentgrass turf (Rothwell 2003;

34 Kostromytska and Koppenhofer 2014). McGraw and Koppenhofer (2009) found that pure stands of creeping bentgrass turf are more tolerant to ABW feeding damage than mixed stands of annual bluegrass and creeping bentgrass turf.

Although recent research suggests that ABW can be found in similar numbers in annual bluegrass and creeping bentgrass turf, this research was conducted exclusively in northeastern

United States. Additionally, the majority of previous research on ABW distribution and host preference is based on the larval stage of the insect with only limited studies investigating the occurrence of adult ABW associated with host plants. In western North Carolina, the adults are the only life stage seen regularly yet damage is still observed. Like the hunting billbug it is plausible that the adult stage is the damaging stage in North Carolina. Additionally, behaviorally research conducted on adult ABW only investigated the spring generation of adults. ABW can undergo 2-3 generations a year, with the last generation occurring in the summer months (August and September). This previous research was conducted more than 1000 km northeast of our study sites and the application of those findings to western North Carolina needs to be compared.

While most golf courses in western North Carolina are primarily creeping bentgrass, many courses in this area have significant contamination of annual bluegrass which can result in changing the management approaches for the entire course. Currently, observations and reports from turfgrass managers on golf courses in western North Carolina indicate that ABW damage is restricted to annual bluegrass turf. However, it is important to better understand the role of potential host plants on ABW distribution, abundant overall turf damage and pest management with information being helpful in developing control strategies for turfgrass managers in this region. Therefore, the objective of this research was to determine if host plant abundance

35 (creeping bentgrass and annual bluegrass) and distribution influences adult abundance in specific sites on golf courses.

Materials and Method

Sampling for ABW adults and quantity of annual bluegrass turf was conducted in 2016 on five golf courses, and three golf courses in 2017. Selection of golf courses for sampling was based on known occurrence of ABW from our mapping survey or confirmation with golf course staff that ABW has been present. Insect sampling using a soap flush was conducted to evaluate presence and abundance of ABW adults as well as sampling of associated turfgrass areas to quantify host plant species composition.

Sampling was conducted on fairways at each of the five golf courses in 2016 Burlingame

Country Club (Transylvania County)(35.11539, -82.98029), Cullasaja Club ( Macon County)

(35.08616, -83.53687), Highland Country Club (Macon County)(35.05254, -83.21732), Wildcat

Country Club (Jackson County) (35.0861, -83.15286), and Cliffs at Walnut Cove (Buncombe

County) (35.46269, -82.60232). Fairways consisted of the shorter mown turfgrass (<1.27 cm) adjacent to the higher mown rough (>3.18cm). Perimeters of fairways were selected where

ABW damage was observed in previous years according to turfgrass superintendents. Fairways consisted of creeping bentgrass with annual bluegrass contamination ranging from 0 to 100 percent within the sampled areas. Counting annual bluegrass presence was conducted in May of

2016 and 2017 which is a time of peak growth for the annual bluegrass in western North

Carolina and therefore making it easier to detect the contamination. A 1 m2 frame containing a grid was used to evaluate the amount of annual bluegrass contamination in creeping bentgrass stands of turfgrass. The grid contained 225 total squares (each square was approximately 6 cm2).

Filament trimmer string was attached to one side of the grid, stretched to the opposite side and

36 tightly fastened to the frame. The frame was constructed of 2.54 cm PVC pipping for each side with scheduled 40 elbow joints to fasten the sides together. Each string was approximately 6 cm from the next string, there was a total of 15 strings per side of frame. This was repeated on the adjacent side of the frame forming squares inside the 1m2 frame (see figure 2.9). The grid was tossed in the perimeter of the fairway in areas where ABW damage was observed in recent years.

The turfgrass area within the frame was assessed for relative encroachment of annual bluegrass in the creeping bentgrass stand by counting the squares containing annual bluegrass. The corners of the perimeter were designated with marking spray paint and flags. Within the marked area a soap water flush was conducted for adult ABWs. The number of spaces in the grid containing annual bluegrass was then divided by 225 and the percent value of contamination was calculated

(# of annual bluegrass/ total number of squares 225 x 100 = % of relative annual bluegrass contamination in the creeping bentgrass stand).

Turfgrass composition was assessed at 30 locations on each of the five courses in 2016.

The sampled areas were revisited monthly with the 30 plots being sampled once a month for adult weevils from May to August. The plot areas did not receive any insecticide during the duration of this study. A soap flush was conducted directly to the turfgrass area within the 1 m2 plot. After applying the soap solution to the turfgrass area, the grass surface was examined closely for ABW presence for three minutes before moving to the next plot. All observed ABW were hand collected from each turfgrass plot, recorded and then placed in vials containing 95% ethyl alcohol and returned to the laboratory. This process was repeated for each of the 30 plots on the golf course.

In 2017, this study was repeated on three golf courses Burlingame Country Club

(Transylvania County)(35.11539,-82.98029), Cullasaja Club (Macon County)(35.08616,-

37 83.53687), and Cliffs at Walnut Cove (Buncombe County)(35.46269,-82,60232). These courses were selected based on observation of the highest levels of adults in 2016 at those locations. The samples were increased to 50 plots per courses to keep the 150 total samples consistent for both years. Turfgrass composition was assessed on 9 May 2017. Sampling was performed using the grid with the exact technique as described above. Plots were revisited every two weeks and were left untreated of any insecticides during the duration of this study. The 50 plots were sampled twice in May, June and July and only once in August.

Results and Discussion

All statistical analysis was carried out using the statistical software package SAS (SAS

Institute, 2001). These data were analyzed by Linear regression in PROC Genmod (distribution= negative binomial) after transformation with log (x + 1). Analysis was used to test for an effect of annual bluegrass on the presence of adult weevils.

The data were separated by year. The annual bluegrass percentages at each course were grouped into three classifications, low infestation (<15% annual bluegrass), medium infestation

(15-30% annual bluegrass), and high infestation (>30% annual bluegrass). The adult ABW populations were averaged in each of the groupings and means were used for comparisons.

2016

All data were collected from five courses in 2016. There was a total of 30 data points for each course totaling 150 data points for each month. Each of the courses had data for the months

May, June, July, and August. The ANOVA test compared the effects of annual bluegrass percentages on the abundance of adult ABW by each month at each course. In the month of June, at Wildcat Cliffs Country Club the mean number of adult ABW decreased as annual bluegrass percentages increase from low (푥̅ = 6.1667) to high (푥̅ = 1.556) (z = 2.72, P<.0066). In August,

38 at Wildcat Cliffs Country Club the data show adult ABW means decreased as annual bluegrass percentages increase from low infestation (푥̅ = 1.083) to high infestation (푥̅ = .111) (z = 2.11,

P<.0352). The trend of decreasing adult ABW populations as the percent of annual bluegrass infestation increased was also observed at Highland County Club. However, only the data collected in the months of July and August show significant P-values (P<0.05) for mean differences. These data suggest that in the month of July adult ABW populations decrease as annual bluegrass percentages increase from low (푥̅ = 16.1) to medium (푥̅ = 3.5) (z = 2.43,

P<.0151). The adult ABW populations decrease as annual bluegrass percentages increase from low (푥̅ = 16.1) to high (푥̅ = 5) (z =2.75, P<.0246). For the month of August at Highland Country

Club these data indicate that the increased percentage of annual bluegrass from low (푥̅ = 1.292) to medium (푥̅ = .033), will result in lower populations of adult ABW (z = 2.27 P<.0234).

2017

All data were collected from three courses. There was a total of 50 plots for each course totaling 150 data points. Golf courses were sampled twice a month and the average was taken to represent each month. The ANOVA test compared the effects of annual bluegrass percentages on the abundance of adult ABW by month at each course. The data collected from Burlingame in the month of July show as annual bluegrass percentages increase from low (푥̅ = 1.4722) to medium (푥̅ = 2.875) there is a significant increase in adult ABW (z = -2.05, P<.0405). This was also observed with data for the month of August, as annual bluegrass percent infestation increases from low (푥̅ = .75) to medium (푥̅ = 1.875), there is a significant increase of adult

ABW. (Z = -2.46, P<.0289). An increase of adult ABW can be seen as the percent of annual bluegrass infestation increases from low (푥̅ = .750) to high (푥̅ =1.8125) (z = -2.24, P<0.0175). At

Cliffs at Walnut Cove, these data show for the month of June there is a decrease of adult ABW

39 as annual bluegrass contamination increased from medium (푥̅ = 2.603) to high (푥̅ = 1.00) (z =

2.31, P<.0321). This is true for data in the month of July as well, there is a decrease in the number of adult ABW as annual bluegrass contamination increases from medium (푥̅ = 3.30) to high (푥̅ = 1.23) (z= 2.06, P<.0399).

These data show that adult ABW populations do not as the contamination of annual bluegrass increases on golf courses in western North Carolina. In 2016, these data indicate that on two courses (Highland Country Club, Wildcat Cliffs Country Club) as the level of contamination with annual bluegrass increases there was a lower number of adult ABW populations. In 2017 these data show an increase in the mean number of adults at one course but a decrease in the mean number of adult at another course. Since the observed relationship of annual bluegrass contamination and ABW abundance has limited statistical significance and was not consistent, no conclusions can be drawn relative to defining high risk areas. These findings are consistent with recent studies from the northeastern United States where the ABW persist as a prominent golf course pest. Rothwell (2003) suggest that ABW can be found in both creeping bentgrass and annual bluegrass in similar numbers. However, many golf course superintendents only see damage to stands of annual bluegrass and not creeping bentgrass. Stands of pure creeping bentgrass are able to tolerate feeding better than mixed stands and pure stands of annual bluegrass (McGraw and Koppenhofer 2010 Kostromytska and Koppenhofer 2014). This could explain why superintendents often see damage to annual bluegrass but not the adjacent creeping bentgrass. This makes proper scouting with soap flushes for this insect pest extremely important for management strategies. The level of annual bluegrass contamination is not a reliable indicator of potentially damaging populations of ABW nor can those sites we targeted as high risks areas for sampling.

40 Literature Cited

Cameron, R.S. 1970. Biology and control of a species of Hyperodes (Coleoptera: Curculionidae), a pest of turfgrass in New York. M.S. thesis. Cornell University, Ithaca, NY.

Cameron, R. S. and N. E. Johnson. 1971. Chemical control of the “annual bluegrass weevil,” Hyperodes sp. nr. anthracinus. J. of Econ. Entomol. 64(3): 689-693.

Rothwell, N. L. 2003. Investigation into Listronotus maculicollis (Coleoptera: Curculionidae), a pest of highly maintained turfgrass. Ph.D. dissertation, University of Massachusetts, Amherst.

Tashiro, H. and R.W. Straub. 1973. Progress in the control of turfgrass weevil, a species of Hyperodes. Down to Earth. 29:8-10

Vittum, P.J. 1980. The biology and ecology of the annual bluegrass weevil, Hyperodes sp. near anthracinus (Dietz) (Coleoptera: Curculionidae). Ph.D. thesis. Cornell Univ., Ithaca, NY. 164

Vittum, P. J., M. G. Villani, and H. Tashiro. 1999. Turfgrass insects of the United States and Canada. Cornell University Press. Ithaca, NY

Figure 2.1 Regression model, X axis percentage of annual bluegrass (poa), Y axis the number of adult weevils observed in the plots across all sampled months in 2016 at course 1

(Cullasaja Club).

Figure 2.2 Regression model, X axis percentage of annual bluegrass (poa), Y axis the number of adult weevils observed in the plots across all sampled months in 2016 at course 2

(Wildcat Cliffs).

Figure 2.3 Regression model, X axis percentage of annual bluegrass (poa), Y axis the number of adult weevils observed in the plots across all sampled months in 2016 at course 3

(Highland Country Club).

Figure 2.4 Regression model, X axis percentage of annual bluegrass (poa), Y axis the number of adult weevils observed in the plots across all sampled months in 2016 at course 4

(Burlingame).

Figure 2.5 Regression model, X axis percentage of annual bluegrass (poa), Y axis the number of adult weevils observed in the plots across all sampled months in 2016 at course 5

(Cliffs at Walnut Cove).

Figure 2.6 Regression model, X axis percentage of annual bluegrass (poa), Y axis the number of adult weevils observed in the plots across all sampled months in 2017 at course 1

(Cullasaja Club).

Figure 2.7 Regression model, X axis percentage of annual bluegrass (poa), Y axis the number of adult weevils observed in the plots across all sampled months in 2017 at course 2

(Burlingame).

Figure 2.8 Regression model, X axis percentage of annual bluegrass (poa), Y axis the number of adult weevils observed in the plots across all sampled months in 2017 at course 3

(Cliffs at Walnut Cove).

Figure 2.9 Weed Grid used to quantify the Composition of annual bluegrass within a plot

50 Table 2.1. 2016 Golf courses and sampling dates

Courses Sampling Dates

Burlingame Country Club 5/23/16 6/27/16 7/18/16 8/2/16 Wildcat Country Club 5/18/16 6/29/16 7/19/16 8/3/16 Cliffs at Walnut Cove 5/31/16 6/30/16 7/20/16 8/4/16 Cullasaja Club 5/10/16 6/28/16 7/18/16 8/2/16 Highlands Country Club 5/18/16 6/29/16 7/19/16 8/3/16

51 Table 2.2. 2017 Golf courses and sampling dates

Courses Sampling Dates Cullasaja Club 5/9/17 5/31/17 6/14/17 6/25/17 7/11/17 7/24/17 8/8/17 Burlingame Country Club 5/9/17 5/31/17 6/16-17/17 6/26/17 7/12/17 7/25/17 8/9/17 Cliffs at Walnut Cove 5/10/17 5/30/17 6/16-17/17 6/26/17 7/11/17 7/25/17 8/9/17

52 Table 2.3. Adult ABW found compared to percent of annual bluegrass (poa) present at each plot, in May, June, July and August 2016.

Poa% May June July August BG 0 1 4 3 1 3 1 1 18 5 5 3 6 0 0 10 4 2 10 0 12 2 0 1 1 13 1 0 1 2 14 1 1 2 0 14 0 0 2 0 15 1 3 0 0 26 2 4 1 0 20 2 0 7 2 22 0 4 0 0 24 0 0 4 1 25 2 2 0 0 28 4 21 25 2 28 1 2 2 0 29 1 7 0 0 30 1 0 0 0 35 1 2 3 1 35 0 1 0 0 36 0 1 0 0 36 0 7 2 0 38 0 27 43 1 41 0 2 1 0 41 1 1 22 6 44 9 2 7 2 55 1 2 2 3 68 2 1 2 1 70 1 23 12 0 86 1 1 4 0 WCC 0 2 7 1 1 1 0 3 0 1 2 0 3 0 1 2 0 4 1 1 4 0 6 3 6 4 0 8 4 0

53 Table 2.3. (Continued) 6 1 8 0 0 8 0 12 3 1 12 0 5 1 0 14 0 5 1 1 14 0 12 0 1 15 2 1 5 0 16 0 16 4 0 17 1 1 1 1 21 0 0 0 0 21 0 5 0 1 22 1 1 5 0 24 0 0 1 0 25 1 0 0 0 27 1 0 0 0 30 4 3 1 1 31 0 1 2 0 33 0 0 4 1 35 1 1 1 0 38 1 0 0 0 40 0 1 7 0 44 0 0 0 0 54 1 8 0 0 64 0 3 1 0 71 1 0 0 0 CWC 0 0 1 2 2 5 0 0 0 0 9 0 0 0 0 10 0 1 1 1 13 0 0 0 1 14 2 0 0 0 14 0 1 0 0 15 0 0 0 0 20 1 0 0 0 23 0 0 1 0 24 0 0 0 0 26 0 1 0 0 26 0 0 0 0 27 1 1 2 0 28 3 0 0 1 28 0 0 0 0 29 0 0 0 0

54 Table 2.3. (Continued) 30 1 0 0 0 30 0 0 0 0 32 1 0 1 1 35 0 1 0 0 38 1 0 0 0 41 0 0 0 0 43 0 0 0 0 45 0 0 0 0 46 0 0 1 0 58 0 0 1 1 67 0 0 0 0 70 0 0 0 1 71 1 0 1 0 CC 2 0 1 2 0 4 0 6 7 0 8 3 12 3 5 9 0 0 0 0 12 2 31 2 4 14 2 7 14 1 14 1 4 1 0 15 2 22 5 0 19 1 26 6 0 20 1 5 11 0 21 1 10 8 0 23 2 3 0 0 24 1 13 17 0 25 2 7 3 0 29 4 16 7 6 30 2 0 12 0 43 1 8 5 1 43 3 63 0 1 46 2 0 1 0 46 1 3 0 1 47 3 1 3 0 48 0 1 4 2 54 1 7 2 0 55 4 53 15 3 61 0 4 14 1 64 0 8 1 2 67 1 14 7 1 78 0 3 12 0

55 Table 2.3. (Continued) 88 0 0 0 0 100 1 3 4 0 HCC 0 2 25 83 16 0 1 20 24 11 0 2 4 1 1 3 3 5 12 0 6 0 6 31 1 6 4 17 9 0 9 3 11 23 1 9 2 4 0 1 12 0 6 0 0 13 1 9 21 1 13 3 12 5 0 14 6 1 2 7 14 2 7 0 1 15 0 44 15 1 16 0 7 4 2 18 5 35 7 0 18 2 5 0 0 21 7 9 2 0 21 1 24 1 0 23 1 9 7 0 32 1 10 1 1 34 3 2 3 3 35 3 33 28 1 38 2 2 5 0 39 3 9 1 1 39 3 11 1 3 40 3 3 5 0 43 4 13 3 0 44 1 16 1 4 55 3 21 2 0

56 Table 2.4. Adult ABW found compared to percent of annual bluegrass (poa) present at each plot, in May, June, July and August 2016.

Poa % May_1 May_2 June_1 June_2 July_1 July_2 August_1 CC 0 0 8 0 0 0 0 0 0 0 1 0 1 0 0 0 0 0 1 0 0 0 1 1 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 2 0 3 0 0 1 0 0 0 0 4 0 0 0 1 0 0 4 4 0 0 0 0 0 0 2 5 0 0 0 0 0 0 0 8 0 0 0 0 0 0 1 10 1 0 0 1 0 2 0 10 0 0 0 0 2 0 1 11 0 1 0 0 0 0 0 11 0 4 0 0 0 0 0 14 0 2 0 0 0 0 0 15 0 1 0 1 0 0 0 16 0 0 0 0 0 0 1 18 0 0 1 0 7 0 1 20 0 0 0 0 0 0 0 22 0 0 0 0 0 0 0 22 1 0 0 0 21 0 1 25 0 0 0 1 0 0 0 27 0 0 0 0 0 0 0 27 0 0 0 0 0 0 0 29 0 1 0 0 0 0 0 29 0 4 1 0 0 0 1 29 0 0 0 0 0 0 0 29 0 7 0 0 0 0 0 29 0 1 0 0 0 0 0 30 0 0 0 0 0 0 0 38 0 7 0 0 0 0 0 38 1 0 0 0 0 0 1 38 0 0 2 0 0 0 0 38 0 0 0 0 3 0 0 41 0 0 0 0 0 0 0 44 1 0 1 0 0 0 0

57 Table 2.4. (Continued) 47 0 1 1 1 0 0 0 50 0 0 0 0 0 0 1 50 0 0 0 0 1 2 0 50 0 0 0 0 0 0 0 50 0 1 0 0 0 0 2 55 0 0 0 0 0 0 0 55 0 0 1 0 1 0 0 64 0 0 1 0 0 0 1 72 0 0 0 1 0 0 2 77 0 0 1 0 0 0 0 90 0 0 0 2 14 0 1 96 1 0 1 0 0 0 0 100 0 0 1 0 0 0 0 BG 0 0 0 1 0 1 0 0 0 0 0 1 1 0 0 0 0 0 0 1 1 0 0 0 0 0 1 1 2 1 0 1 0 0 0 1 1 0 0 3 0 0 1 1 3 1 0 0 0 0 0 1 2 0 0 0 3 1 0 0 0 1 0 0 3 0 0 0 0 1 0 0 5 8 1 3 1 0 0 14 6 0 0 1 3 10 0 0 7 0 2 1 2 2 1 2 11 0 0 1 5 0 2 2 12 0 0 3 2 0 0 3 14 2 0 1 0 2 1 1 14 0 0 2 0 2 0 0 15 2 0 0 0 0 0 1 15 0 1 11 0 1 1 2 16 0 0 1 0 1 0 1 17 0 0 3 0 1 0 0 18 0 0 1 5 1 0 6 18 4 1 1 1 9 0 2 18 0 2 1 1 2 0 2 21 0 0 2 9 4 1 4 22 3 0 1 0 2 1 1 24 0 4 7 2 1 0 1 24 2 0 0 0 1 1 0 24 0 1 5 3 1 2 6

58 Table 2.4. (Continued) 27 5 7 2 0 2 0 2 27 3 2 0 6 16 0 11 28 1 1 2 10 2 4 8 28 2 0 8 15 4 0 0 30 0 0 5 1 2 1 0 30 0 0 0 0 1 0 2 60 1 2 3 13 13 0 0 63 2 2 1 0 1 0 1 63 2 0 0 0 1 0 0 67 0 1 8 1 1 1 11 72 0 1 0 1 1 0 5 72 0 1 6 0 1 0 2 75 0 1 2 0 1 0 9 79 2 2 0 7 1 3 5 81 0 3 2 1 5 0 2 89 0 0 1 2 8 1 3 92 0 0 1 0 3 1 7 94 0 3 5 2 3 3 1 96 1 0 0 1 3 0 2 100 0 1 4 1 2 1 1 100 0 0 1 0 1 0 0 100 0 0 7 0 1 2 1 CWC 0 0 0 1 4 6 0 0 0 0 0 1 1 6 0 4 0 3 1 1 1 3 1 2 0 0 1 0 2 2 0 2 0 0 3 1 0 5 1 1 0 0 1 1 1 1 0 0 0 4 6 1 1 1 0 2 0 0 1 2 0 2 0 0 1 0 1 2 4 0 8 2 2 0 0 1 5 3 4 3 2 0 0 1 3 1 2 4 4 0 1 0 3 1 2 8 5 0 2 0 1 2 2 0 5 0 1 0 3 0 1 0 7 0 1 0 1 0 0 2 7 0 1 1 0 1 1 4 8 0 1 1 2 0 0 0 8 0 1 1 4 23 2 1 8 0 1 1 1 1 2 6

59 Table 2.4. (Continued) 11 0 0 1 6 1 2 0 14 1 3 0 0 0 0 0 15 0 4 2 6 7 2 8 17 0 3 2 3 14 8 0 19 0 0 1 5 20 1 4 19 0 0 3 3 3 0 1 19 0 0 2 3 4 0 3 21 0 1 0 2 2 0 2 22 0 0 0 0 0 0 0 22 0 0 0 0 0 0 0 22 0 0 0 0 1 0 0 22 0 0 1 4 2 2 3 28 0 2 1 5 3 0 2 28 0 1 0 0 0 0 1 29 1 0 1 7 23 4 11 29 1 0 1 3 1 2 3 30 0 0 1 6 4 1 3 30 0 0 2 5 2 2 0 33 0 1 1 1 3 0 2 34 0 0 1 5 0 0 1 36 0 0 0 1 6 0 0 40 0 1 1 5 18 0 7 41 0 1 0 0 0 0 1 42 0 1 1 0 0 0 0 42 0 0 1 0 0 2 0 44 0 0 1 0 1 0 1 44 0 2 0 2 0 1 3 44 0 1 2 2 0 0 2 44 0 4 1 0 0 0 11 51 0 9 0 0 0 0 14 51 0 1 1 0 1 0 0

60 APPENDICES

61 APPENDIX A

Survival of annual bluegrass weevil larvae, Listronotus maculicollis, on turfgrasses found in

western North Carolina

The annual bluegrass weevil (ABW), Listronotus maculicollis Kirby (Coleoptera:

Curculionidae), is a serious and expanding pest on golf courses in eastern North America. With

ABW resistance to insecticides being a problem, different management strategies need to be addressed. ABW was first discovered causing damage on golf courses located in western North

Carolina in 2006. Creeping bentgrass (Agrostis stolonifera L.) is the dominant grass species on most golf courses located in western North Carolina (B.T Corbett personal observation).

However, areas with heavy damage from ABW often are contaminated with annual bluegrass, a highly invasive weed (Poa annua L). Annual bluegrass is highly susceptible to ABW damage and recent United States Golf Association (USGA) research conducted in the northeastern

United States reported damage to swards of creeping bentgrass from ABW. Since North Carolina is in the transition zone for management of growing turfgrass, some courses have converted fairways to warm season hybrid bermuda grass (Cynodon dactylon x Cynodon transvaalensis) turf cultivars. Golf courses with newly-converted fairways have often reported ABW damage to the turf on putting greens and tees (consisting of cool season grass), but no damage on warm season fairways. In this study, the survivability of ABW larvae on three different turfgrasses, creeping bentgrass (Agrostis stolonifera L.), annual bluegrass (Poa annua), and hybrid bermuda were evaluated (Cynodon dactylon x Cynodon transvaalensis), Turfgrass tolerance (damage to grass), antibiosis (larval survival and growth) and antixenosis (inability of a plant to serve as host) were looked at in this study for ABW feeding preference and the number of eggs

62 oviposited. There were no differences in ABW survival and feeding found among any of the different turfgrasses. No eggs or larvae were found during the experiment.

63 Introduction

The annual bluegrass weevil (ABW), Listronotus maculicollis Kirby, is a severe pest of highly maintained short mown turf on golf courses in North America (Vittum 1999). This weevil is a pest on golf courses expanding from northeastern United States and into provinces of Canada

(McGraw and Koppenhofer 2016, Vittum 1999, Simard 2007). ABW was first discovered causing damage on golf courses in 1931 in Connecticut. Since 1931 it has been confirmed in 14 states with its origin in the metropolitan New York City area (McGraw and Koppenhofer 2016).

Since 2008 many states located south of New York have reported damage from ABW (McGraw and Koppenhofer 2016).

ABW complete two to three generations per year in the northeastern United States. The first generation typically causes the most severe damage to turfgrass on golf courses in this area

(Vittum 1999). Adults emerge from overwintering sites located adjacent to golf course fairways.

These sites typically consist of mixed leaf litter, tall grasses, pine straw and mulch. The adults migrate toward the shorter mown turfgrass where adult females chew notches in the grass stem, and oviposit eggs inside the sheath of the turfgrass plant. The larvae that emerge develop through five instars. The first to third instar larvae are stem borers causing some damage to the turfgrass plant, while the most severe damage is caused by the fourth and fifth instar larvae. Once they develop to the final instars, larvae drop into the soil and begin feeding on the roots and meristem of the turf plant. This feeding is detrimental to the turfgrass plant. A single larva can destroy between 12 to 20 turfgrass stems (Cameron and Johnson 1971). When densities exceed 100 larvae/0.1m2, damage is severe visually and in playability of the turf (McGraw 2016). Adults can also feed on the grass creating notches in the turfgrass blades, weakening the grass, but since this is above the meristem, it causes no severe damage (Cameron and Johnson 1971).

64 The only effective control for ABW is chemical control (Vittum 1999). The intensive reliance on synthetic pesticides for the past 40 years with multiple applications (six to ten applications per year) has led to insecticide resistance for most pyrethroid insecticides (Cowles et al. 2008). Currently, newer insecticide chemistries can provide effective alternative to pyrethroids; however, the repetitive use of these products may also result in resistance (McGraw

2016).

Host plant resistance has been investigated as a possible pest management tool for ABW control. It has been successfully implemented for insect management for various pests in many different cropping systems. Warm season turfgrass varieties have been evaluated for their resistance to a variety of phytophagous pests (Quisenberry 1990; Reinert et al., 2004; Shortman et al., 2002 Marshall et al., 2015) while laboratory studies demonstrate that some warm season grass cultivars exhibit high levels of antibiosis on fall armyworm (Spodoptera frugiperda) larvae

(Braman et al., 2000). Braman et al. (2000) published data that indicates two varieties of zoysiagrass turfgrasses had moderate resistance to mole cricket damage in comparison to other species of zoysiagrass (Braman et al., 2000). Other warm season varieties such as Raleigh St.

Augustine turfgrass, possess high resistance to southern chinch bugs (Blissus insularis) in

Florida (Anderson et al., 2006).

ABW damage typically occurs in areas contaminated with high levels of annual bluegrass. However, larval density was similar in plots with pure annual bluegrass and pure creeping bentgrass, demonstrating that larval density is not determined by the turfgrass species indicating this pest can survive on both species (Rothwell 2003). However, pure stands of creeping bentgrass are more tolerant to ABW larval feeding than mixed stands of annual bluegrass and creeping bentgrass. There is no effect of host species on spring adult and larval

65 distribution (McGraw and Koppenhofer, 2009). Studies by Kostromystka (2014) found significant differences in the number of eggs oviposited, the number of larvae that hatched, the overall fitness of the larvae and the number of larvae that successfully pupated on different cultivars of bentgrass. Data show that ABW adult females preferred annual bluegrass for oviposition compared to creeping bentgrass (Kostromystka 2014).

The goal of this study is to compare ABW survival on hybrid bermudagrass to known host plant, creeping bentgrass and annual bluegrass. Along with looking at ABW survival in a warm season turfgrass as this has not been previously documented.

Material and Methods

INSECTS

ABW populations collected from each generation were used in the experiment: (1) adults emerging from overwintering sites in the spring, overwintered generation adults were collected;

(2) Spring-generation ABW collected in late May; (3) Summer generation ABW collected in mid to late June. All weevils were collected from Headwaters Golf Course (Jackson

County)(35.08572, -83.0728) Adults were hand collected from tee boxes and fairways on the golf course. Overwintered adults were collected 30 March, 4 April and 10 April. The spring generation was collected 2 May, 9 May and 16 May. The summer generation adults were collected 13 July, 15 July and 20 July. Adults were sexed under 10 x microscope using Cameron and Johnson (1971) taxonomic and ID guide. Adults were kept in 840-mL plastic containers filled with turfgrass clippings and a moist paper towel and stored at room temperature (23 C).

Weevils were not held in containers for more than 24 hours.

PLANT MATERIAL

66 Three different turfgrasses were used for the experiment, creeping bentgrass (Penncross), annual bluegrass and hybrid bermudagrass (Tifway 419). These turfgrasses are the most common turfgrasses found on fairways in western North Carolina where ABW damage is observed.

Creeping bentgrass used for the experiment was grown from plugs taken from a local sod farm (Turf Mountain Sod, Morgantown, NC, USA). All turfgrass plugs were taken in 2017 using

Accuform Turf Plugger (PARAIDE, 6 cm diameter). Plugs for overwintered generation adults were taken 9 February, 7 March for spring generation and 18 May for summer generations, plugs were prepped and placed in the greenhouse. Hybrid bermudagrass plugs were taken from Lake

Wheeler Turf Research Lab (Raleigh, NC, USA). Annual bluegrass plugs were taken from

Roaring Gap Golf Club (Roaring Gap, NC, USA). Plugs approximately 6.35 cm in depth were taken from areas that have had no history of ABW and had not been treated with any insecticides for the previous 18 months. Grass plug roots were washed free of soil with water before planting.

Plugs were propagated for at least three weeks before they were used in experiments. All grasses were grown in 540-mL deli cups with drainage holes and mesh placed over the drainage hole to prevent ABW from escaping. The cups were filled with a mixture of a pasteurized for 1 h at 120

C, the potting mix (Echo Horticulture sunshine mix 3) was made up of 70-80% Canadian

Sphagnum, dolomitic lime, and wetting agent and play sand (Quikrete) ratio 2/3 potting soil to

1/3 play sand. Plants were fertilized weekly with 10-10-10 NPK; (The Scotts Miracle-Gro,

Marysville, OH, USA), and watered as necessary. Plants were clipped as needed to maintain fairway height of 1.3 cm. All turfgrass plugs were grown in the greenhouse at 17-25 C for the duration of the experiment March-September 2017.

LARVAL SURVIVAL, GROWTH, DEVELOPMENT AND GRASS TOLERANCE

67 ‘Penncross’ creeping bentgrass, ‘Tifway 419’ hybrid bermudagrass and annual bluegrass were tested for larvae survival, larvae development and host tolerance for at least seven days in the greenhouse in 540-mL plastic containers with drainage holes and mesh covering the bottom.

Overwintering adults (5 males and 5 females) were introduced into each container, and the containers were covered with a screened lid and randomly arranged in the greenhouse (Turfgrass plugs were arranged using random experimental generator ARM) (see figure 3.3). Selected turfgrass plugs were removed from greenhouses in seven day intervals and subsequently examined for eggs and larval presence (7, 14, 21 and 28d). There were four replications for each turfgrass variety for 7, 14, 21 and 28d and the study repeated three times in March-April 2017.

This process was also repeated for the spring and summer generations (four replications and three experimental runs) The overwintered adults were introduced 31 March, 5 April and 11

April. The spring generation adults were introduced 3 May, 10 May and 17 May. The summer generation adults were introduced 14 July 16 July and 21 July.

Cores selected for evaluation for larvae were placed on a tray and teased apart by hand, dividing horizontally into halves. Both top and bottom of turfgrass cores were thoroughly searched before placing turfgrass core fragments in a lukewarm (30 C) salt water solution (950 mL of salt per 3.78 L of water). Salt in the solution irritates larvae causing them to float to the top of the water. All floating material was examined by the aid of a hand lens and a dissecting microscope. This extraction method was only used for the “overwintered” generation. The spring and summer generations test plugs were removed from the greenhouse and cores were externally examined for presence of larvae then divided horizontally into halves. The top and bottom of the turfgrass cores were manually searched for ABW life stages; the top section was then placed upside down in a Berlese funnel. A 20-watt light bulb was placed over the funnel for

68 the heat source. A screen was placed in the bottom of the cylinder inside the funnel and a jar containing 95% ethanol was screwed on below the funnel. The funnels and cores were placed in a heated room (30C) for seven days and jars were checked every day for larvae. The room had supplemental lighting programmed for 12h of darkness and 12h of light each day.

OVIPOSITION AND EGG SURVIVAL

On 9 August 2017 adult ABWs were manually collected at night from Headwaters Golf

Course and the adults were kept in plastic 540-mL deli cups at ambient temperature (10 C) until introduced into plastic laboratory containers the next morning. Adults were sexed using a dissection microscope (10X) and 1 male and 1 female were placed in a 59-mL plastic container containing annual bluegrass clippings collected from NC State Turf Research Station for creeping bentgrass greens with no history of ABW. A total of 100 containers were used and each of the plastic containers were sealed with mesh lids preventing adults from escaping. The plastic containers were kept at 25 C and 50% humidity. The plastic cups were visually inspected daily for 35 days. This was repeated on 14 August 2017, with Lepidoptera diet donated from Shawnee

Gundry (McMorran 1965) to look at multiple diets to promote egg laying.

69 EGG SURVIVAL

On 27 July 2017 100 adult females ABW were manually collected at night from

Headwaters Golf Course, Cashiers, NC, USA. ABW were kept in plastic 540-mL deli cups at ambient temperature (10 C) until dissection the following morning in Raleigh, North Carolina at the North Carolina State University Lake Wheeler Turfgrass Research Center. Females were dissected under a dissecting microscope and eggs removed from the adults were placed on moist filter paper placed in petri dishes (5cm diameter). 73 eggs were placed on to petri dishes with each dish containing 10 eggs, except for one that only contained three eggs. Eggs were maintained at 25 C and 70% humidity. The eggs were checked daily and remained in this environment for the duration of 10 days checking for emerging larval. For the duration of the experiment, eggs were held at optimal temperature (20-30 C) and given adequate moisture for egg development.

Results and Discussion

LARVAL SURVIVAL, GROWTH, DEVELOPMENT AND GRASS TOLERANCE

No larvae or eggs recovered from the turfgrass plugs for the duration of this experiment. .

Studies that introduce subterranean pest into artificial arenas have resulted in similar conclusions; white grub larvae also have been reported to experience poor recovery when relocated for damage threshold studies in laboratory and greenhouse settings (Potter 1982).

Research conducted on the hunting billbug, Sphenophorus venatus vestitus, in North Carolina also concluded similar results. The hunting billbug and the ABW are similar species that are often mistaken as one another on golf courses in western North Carolina (B.T Corbett personal communication). Doskocil and Brandenburg (2012) found in the initial biological studies of this pest that the adult hunting billbug, not the larvae, caused damage to the turfgrass in containers

70 placed in the field. The authors also reported that the containers which encased adults yielded no larvae at the conclusion of the experiment (Doskocil and Brandenburg 2012) which reiterates the challenges of finding larvae from some beetle species. There was no evidence of parasitism or disease present and the basis for no larvae in the experiment was unclear.

The peak time for ABW in western North Carolina was May through August for 2016 and 2017. ABW has a complete life cycle with 1-3 generations per year in western North

Carolina. Damage from ABW is typically seen in late May to early June and again in early

August, yet larvae are rarely found on golf courses during this time. All adults were collected when peak quantities were observed on the golf courses for the experiment; however, no larvae were found in any of the greenhouse plugs. While most research in the northeastern United

States is conducted on larvae, we failed to find any in our research trails. The purpose of this study was to see if different types of turfgrasses would influence the overall fitness and number of larvae present. Since no larvae were recovered we cannot conclude which variety of turfgrass was more or less desirable for larval survival and susceptibility to feeding damage. Contributing factors for the lack of success of this experiment could be: the adults were not left in the containers long enough to mate and oviposit fertile eggs, not enough adults were introduced in each container to find suitable mates and fertile females, some adults could have escaped the containers, the containers did not provide a suitable breeding environment or the overall health and fitness of the adults may have been compromised during collection, sexing, transportation and introduction of ABW into grass plugs. It is still unclear if the larval of ABW is the damaging life stage in western North Carolina and it also remains unclear on how different turfgrass species influence the overall survival of ABW and if ABW can survive in hybrid bermudagrass in western North Carolina.

71 OVIPOSITION AND EGG SURVIVAL

A total of four eggs were recovered from adults placed in the 59-mL plastic containers.

All eggs were recovered from a single container that contained Lepidoptera diet. The other 99 containers yielded zero eggs. Eighty-one percent of all adults that were placed in the Lepidoptera diet-filled containers for 35 days remained alive and appeared healthy. Only 43% of the adults remained alive for the duration of the experiment that was placed in the 2 oz plastic containers filled with clippings of annual bluegrass. There were no eggs recovered from any of these containers. Adult females also may have not preferred to lay eggs in artificial areas as this has been seen in other Coleoptera insects such as hunting billbugs (Doskocil and Brandenburg 2012).

Of the 73 eggs that were manually removed from females and placed on to Petri dishes, no eggs hatched or appeared close to hatching after 10 days. Vittum (1980) indicates that ABW eggs hatch within 5-7 days. All eggs were checked daily and moisture was added to the filter paper. Dembilo et al (2011) stated that temperature is the main abiotic factor influencing biology, ecology, and population dynamics of poikilothermic organisms such as arthropods based on his work on R. ferrugineus, the Red Palm weevil. Dembilo, found that this weevil species would have successful egg development in temperatures above 13 C. During the duration of our experiment the temperature was always about 13 C, which is optimal for egg development.

Vittum (1980) research indicates that temperatures between 20-30 C were optimal for larvae development. Vittum (1980) studies show that as you increase to 30 C, the developmental time for the larvae decreases compared to lower temperature. During the duration of our experiment, eggs were held at optimal temperature for egg hatch and embryos development. It is possible that eggs collected from females were not fertilized by males on the golf courses, it is also possible that the removal of these eggs by dissection caused the eggs to no longer be fertile.

72 Literature Cited

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Braman, S.K., R.R. Duncan, and M.C. Engelke. 2000. Evaluation of turfgrasses for resistance to fall armyworms (Lepidoptera: Noctuidae). HortScience 35:1268–1270.

Cameron, R. S. and N. E. Johnson. 1971. Chemical control of the “annual bluegrass weevil,” Hyperodes sp. nr. anthracinus. J. of Econ. Entomol. 64(3): 689-693.

Cowles, R.S., A.M. Koppenhöfer, B.A. McGraw, S.R. Alm, D. Ramoutar, D.C. Peck, P. Vittum, P. Heller, and S. Swier. 2008. Insights into managing the annualbluegrass weevils. Golf Course Manage. August: 86−92.

Dembilo. O, Tapia, G.V. Tellez, M.M, and Jacas J.A. 2011. Lower temperature thresholds for oviposition and egg hatching of the Red Palm Weevil, Rhynchophorus ferrugineus (Coleoptera: Curculionidae), in a Mediterranean climate. Bull Entomol Research. 97-102.

Doskocil, J.P. and Brandenburg R.L. 2012. Hunting Billbug (Coleoptera: Curculionidea) Life Cycle and Damaging Life Stage in North Carolina, With Notes on Other Billbug Species Abundance. J. Econ. Entomol 106: 2045-2051.

Marshall, S. Orr. D. Bradley, L. and Moorman C. 2015. A Review of Organic Lawn Care Practices and Policies in North America and the Implications of Lawn Plant Diversity and Insect Pest Management. HortTechnology 25(4): 437-446.

McMorran A. 1965. A synthetic diet for the spruce budworm, Choristoneura fumiferana (Clem.) (Lepidoptera: Tortricidae). Can. Entomol. 97: 58–62.

73 McGraw, B. A. 2009. The Ecology and the Biological Control of the Annual Bluegrass Weevil, Listronotus maculicollis Kirby (Coleoptera: Curculionidae) Using Entomopathogenic Nematodes (Rhabditida: Steinernematidae and Heterorhabditidae). Ph. D. dissertation, Rutgers, The State University of New Jersey, New Brunswick.

McMorran A. 1965. A synthetic diet for the spruce budworm, Choristoneura fumiferana (Clem.) (Lepidoptera: Tortricidae). Can. Entomol. 97: 58–62.

Potter D. A. 1982. Influence of feeding by grubs of the southern masked chafer on quality and yield of Kentucky bluegrass. J. Econ. Entomol. 75: 21–24.

Quisenberry, S.S. 1990. Plant resistance to insects and mites in forage and turf grasses. Fla. Entomol. 73:411–421.

Reinert, J.A., M.C. Engelke, and J.C. Read. 2004. Host resistance to insects and mites-a major IPM strategy in turfgrass culture. Acta Hort. 661:463–486.

Rothwell, N. L. 2003. Investigation into Listronotus maculicollis (Coleoptera: Curculionidae), a pest of highly maintained turfgrass. Ph.D. dissertation, University of Massachusetts, Amherst.

Shortman, S.L., S.K. Braman, R.R. Duncan, W.W. Hanna, and M.C. Engelke. 2002. Evaluation of turfgrass species and varieties for potential resistance to twolined spittle- bug, Prosapie bicincta (Say) (Homoptera: Cercopidae). J. Econ. Entomol. 95:478– 486.

Vittum, P.J. 1980. The biology and ecology of the annual bluegrass weevil, Hyperodes sp. near anthracinus (Dietz) (Coleoptera: Curculionidae). Ph.D. thesis. Cornell Univ., Ithaca, NY. 164

Vittum, P. J., M. G. Villani, and H. Tashiro. 1999. Turfgrass insects of the United States and Canada. Cornell University Press. Ithaca, NY

Figure 3.1 Arrangement of grasses in greenhouse

75 Table 3.1. Grass arrangement for overwintered trial

Crop Treatment DAT Rep1 Rep2 Rep3 Rep4 Run 1 Hybird bermudagrass 7 106 212 308 405 Hybird bermudagrass 14 112 204 301 409 Hybird bermudagrass 21 101 207 303 412 Hybird bermudagrass 28 109 206 312 403 Creeping bentgrass 7 111 201 309 410 Creeping bentgrass 14 108 211 307 408 Creeping bentgrass 21 105 203 305 407 Creeping bentgrass 28 104 202 311 401 Annual Bluegrass 7 107 210 304 402 Annual Bluegrass 14 102 208 310 404 Annual Bluegrass 21 103 209 302 406 Annual Bluegrass 28 110 205 306 411 Run 2 Hybrid bermudagrass 7 110 205 303 409 Hybrid bermudagrass 14 102 210 301 407 Hybrid bermudagrass 21 112 206 304 405 Hybrid bermudagrass 28 103 212 309 410 Creeping bentgrass 7 104 208 311 404 Creeping bentgrass 14 108 204 307 411 Creeping bentgrass 21 106 209 305 401 Creeping bentgrass 28 101 202 312 408 Annual bluegrass 7 111 203 306 403 Annual bluegrass 14 109 211 310 406 Annual bluegrass 21 107 201 308 402 Annual bluegrass 28 105 207 302 412 Run 3 Hybrid bermudagrass 7 110 203 301 409 Hybrid bermudagrass 14 112 205 306 402 Hybrid bermudagrass 21 101 210 308 410 Hybrid bermudagrass 28 106 209 312 406 Creeeping bentgrass 7 108 204 310 412 Creeeping bentgrass 14 102 208 309 404 Creeeping bentgrass 21 107 211 305 407 Creeeping bentgrass 28 111 206 304 405 Annual Bluegrass 7 105 212 307 403 Annual Bluegrass 14 104 207 303 401 Annual Bluegrass 21 109 202 311 408 Annual Bluegrass 28 103 201 302 411

76 Table 3.2. Grass arrangement of spring generation trial

Crop Treatment DAT Rep1 Rep2 Rep3 Rep4 Run 1 Hybird bermudagrass 7 101 203 311 403 Hybird bermudagrass 14 102 207 303 408 Hybird bermudagrass 21 103 204 308 401 Hybird bermudagrass 28 104 211 307 409 Creeping bentgrass 7 105 212 301 407 Creeping bentgrass 14 106 205 309 402 Creeping bentgrass 21 107 208 302 410 Creeping bentgrass 28 108 201 310 405 Annual Bluegrass 7 109 202 312 411 Annual Bluegrass 14 110 206 305 412 Annual Bluegrass 21 111 209 306 404 Annual Bluegrass 28 112 210 304 406 Run 2 Hybrid bermudagrass 7 101 210 307 405 Hybrid bermudagrass 14 102 212 302 407 Hybrid bermudagrass 21 103 202 301 409 Hybrid bermudagrass 28 104 211 304 402 Creeping bentgrass 7 105 203 306 403 Creeping bentgrass 14 106 208 303 412 Creeping bentgrass 21 107 209 311 404 Creeping bentgrass 28 108 204 309 411 Annual bluegrass 7 109 205 308 410 Annual bluegrass 14 110 201 310 401 Annual bluegrass 21 111 207 312 408 Annual bluegrass 28 112 206 305 406 Run 3 Hybrid bermudagrass 7 101 204 306 410 Hybrid bermudagrass 14 102 210 309 403 Hybrid bermudagrass 21 103 206 305 411 Hybrid bermudagrass 28 104 201 307 401 Creeeping bentgrass 7 105 203 302 407 Creeeping bentgrass 14 106 202 303 408 Creeeping bentgrass 21 107 205 304 412 Creeeping bentgrass 28 108 212 310 402 Annual Bluegrass 7 109 208 311 404 Annual Bluegrass 14 110 211 301 409 Annual Bluegrass 21 111 209 312 406 Annual Bluegrass 28 112 207 308 405

77 Table 3.3. Grass arrangement of summer generation trial

Crop Treatment DAT Rep1 Rep2 Rep3 Rep4 Run 1 Hybird bermudagrass 7 102 212 301 407 Hybird bermudagrass 14 103 205 308 409 Hybird bermudagrass 21 105 206 310 403 Hybird bermudagrass 28 112 211 309 408 Creeping bentgrass 7 104 210 303 405 Creeping bentgrass 14 103 204 311 411 Creeping bentgrass 21 112 203 312 404 Creeping bentgrass 28 111 207 307 412 Annual Bluegrass 7 107 209 306 402 Annual Bluegrass 14 101 201 302 401 Annual Bluegrass 21 110 202 305 410 Annual Bluegrass 28 106 208 304 406 Run 2 Hybrid bermudagrass 7 104 211 305 411 Hybrid bermudagrass 14 111 201 311 405 Hybrid bermudagrass 21 109 209 310 406 Hybrid bermudagrass 28 112 212 308 402 Creeping bentgrass 7 110 210 301 408 Creeping bentgrass 14 108 204 309 407 Creeping bentgrass 21 107 205 305 412 Creeping bentgrass 28 105 206 307 403 Annual bluegrass 7 108 202 302 409 Annual bluegrass 14 101 207 306 404 Annual bluegrass 21 107 208 312 401 Annual bluegrass 28 106 203 303 410 Run 3 Hybrid bermudagrass 7 105 206 311 407 Hybrid bermudagrass 14 102 202 302 404 Hybrid bermudagrass 21 101 209 304 401 Hybrid bermudagrass 28 106 207 303 403 Creeeping bentgrass 7 110 203 306 406 Creeeping bentgrass 14 108 212 309 411 Creeeping bentgrass 21 111 211 301 408 Creeeping bentgrass 28 112 209 307 405 Annual Bluegrass 7 107 201 312 409 Annual Bluegrass 14 103 212 305 410 Annual Bluegrass 21 109 204 312 412 Annual Bluegrass 28 104 208 310 402

78 APPENDIX B

Taxonomic and the Genetic Study of the Annual Bluegrass Weevil

The taxonomy and classification of the ABW has been under much debate since the discovery of this pest in 1931. For 50 years the proper taxonomic identification of this insect between Hyperodes maculicollis and Hyperodes anthracinus was uncertain. It was in 1981 that

Charles O’Brien (O’Brien 1981) identified this insect as Listronotus maculicollis and this publication effectively finalized the classification. Since 1985, all publications on the ABW has identified the insect in the genus Listronotus and species maculicollis.

The annual bluegrass weevil (ABW), Listronotus maculicollis, formally Hyperodes, is a pest of golf courses in the northeastern United States. It was first noticed damaging turfgrass in western North Carolina in 2006 (B.T Corbett personal communication). The initial research on the ABW in western North Carolina was conducted in 2015. Dr. Billeisen, from North Carolina

State University conducted the first biology and ecology studies on the ABW in western North

Carolina and in February of 2016, Bret Corbett (M.S. entomology graduate student) and Dr.

Billeisen sent specimens collected from Wade Hampton (Jackson County) (35.0865, -83.07058) to the North Carolina Plant Disease and Insect Clinic where Dr. Mathew Bertone classified the specimens.

Dr. Bertone identified these specimens as Listronotus maculicollis. Dr. Bertone noticed phenological difference and was unsure of the classification. He photographed the specimens and collaborated with Dr. Charles O’ Brien. In March 2016 Dr. O’Brien identified the specimens as

L. anthracinus. Dr. O’Brien stated “The species of the smaller Listronotus (formerly Hyperodes) can be very difficult to ID. However, maculicollis is only superficially similar to anthracinus.

The former differs from your illustration as follows: the elytral surface is roughly granulate

79 under relatively dense scales and suberect to recumbent setae. It has scattered small iridescent scales especially near the suture and on the declivity. The pronotal carina is less distinct, finer and often incomplete. The suberect setae are denser and much finer.” Dr. Billeisen reached out to Dr. Ben McGraw (Professor of Entomology at Pennsylvania State University) for specimens from Pennsylvania for comparison.

In April 2016, Dr. Bertone received specimens from Pennsylvania along with more specimens from additional locations in western North Carolina (Buncombe County, North

Carolina, Avery County, North Carolina). Upon further identification and comparison to a L. maculicollis specimen in the North Carolina State University Insect Museum, Raleigh, North

Carolina, USA, Dr. Bertone noted that the specimens from Pennsylvania appeared to be closer to

L. anthracinus. Dr. Bertone reached out and sent photos of the North Carolina specimens as well as the Pennsylvania specimens to Dr. Lourdes Charmorro, (Research Entomologist/Curator of

Curculionidae c/o Smithsonian Institution - National Museum of Natural History). Dr.

Charmorro identified seven pictured specimens, three from North Carolina (counties listed above) and four specimens from Pennsylvania all as L. maculicollis. All specimens were sent to

Dr. Charmorro for identification in late April 2016. Dr. Charrmorro and Dr. O’Brien identified all specimens using DNA as L. anthracinus.

In May and June 2016, Corbett reached out to additional states for specimens. Corbett received specimens from Delaware, Virginia, Massachusetts, as well as additional sites in western North Carolina. Corbett and Dr. Bertone reached out to Dr. Brian Weigman (Professor of Entomology, NCSU) to examine genetics(DNA) on specimens received. This is the first genetic sequencing on the Listronotus species in question recorded. The specimens were sequenced using the partial CO1 (barcode region) LC01490f, TY-J-1460, and HC02198r. All

80 base pairs were identical for all specimens. All specimens were identified as L. anthracinus by

Dr. Bertone.

In January 2017, all samples were sequenced at North Carolina State University by Dr.

Weigman were sent to Dr. Charrmorro. Additional samples from North Carolina, Virginia,

Michigan, and Canada (Toronto) were also sent to Dr. Charrmorro. In January 2017, Dr.

Charrmorro sequenced CO1 and IST2 for 96 specimens that were sent in by Corbett and Dr.

Billeisen. All specimens from each location were genetically identical. To date there are specimens from North Carolina, New Jersey, Virginia, Rhode Island, Pennsylvania,

Massachusetts, Michigan, Delaware, New Hampshire, and Canada (Toronto). DNA sequenced from all specimens was genetically the same and appear to be L. maculicollis.

81 Literature Cited

O’Brien, C.W. 1981. The larger (4.5mm+) Listronotus of America north of Mexico (Cylindrorhininae, Curculionidae, Coleoptera), Trans. Am. Entomol. Soc. 107: 69- 123.