BILLBUG (Sphenophorus spp.) COMPOSITION, ABUNDANCE, SEASONAL ACTIVITY, DEVELOPMENT TIME, CULTIVAR PREFERENCE, AND RESPONSE TO ENDOPHYTIC RYEGRASS IN FLORIDA
By
TA-I HUANG
A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE
UNIVERSITY OF FLORIDA
2008
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© 2008 Ta-I Huang
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To my dear parents
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ACKNOWLEDGMENTS
I sincerely thank Dr. Eileen A. Buss, my supervisory committee chair, for her support and guidance and for giving me an opportunity for my professional and personal growth. She has been a role model of professional honesty, work ethic, enthusiasm and dedication balanced with love and care in the personal life, which will inspire me throughout my further career. Working with Dr. Buss was enjoyable and rewarding. I want to acknowledge my supervisory committee members (Dr. Marc Branham and Dr. Kevin Kenworthy) for their contribution. I am grateful for the research sites, assistance and cooperation provided by Mark Kann (University of Florida
Plant Science Unit in Citra, FL), Michael Rowe (Gainesville Golf and Country Club), Mark
Dickson (WestEnd Country Club), Frank Sbarro (LaGorce Country Club), Sean Anderson (Card
Sound Golf Club), Dr. Leah Brilman from Seed Research of Oregon who provided me with seed product, and the Scotts Company provided me the ryegrass seeds. I want to thank all of my lab partners who helped me with my research: Cara Vazquez, Olga Kostromytska, Jessica Platt, Paul
Ruppert, Jade Cash, Megan Gilbert, and Rachel Sheahanand. I also would like to thank Dr.
Michael Thomas (Division of Plant Industries) and Lyle Buss (University of Florida) for their help.
I am grateful for the funding provided by the Florida Turfgrass Association, Florida Golf
Course Superintendents Association, and Golf Course Superintendents Association of America.
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TABLE OF CONTENTS
Page
ACKNOWLEDGMENTS ...... 4
LIST OF FIGURES ...... 8
ABSTRACT...... 9
CHAPTER
1 INTRODUCTION ...... 11
Warm Season Grasses in North America ...... 11 Common Arthropod Pests of Bermudagrass ...... 11 Billbugs Sphenophorus spp. in the United States...... 12 Sphenophorus venatus vestitus in the Southeastern United States ...... 13 Billbug Integrated Pest Management...... 14
2 BIOLOGY AND SEASONAL PHENOLOGY OF BILLBUGS, Sphenophorus spp., IN FLORIDA...... 16
Materials and Methods ...... 17 Composition, Abundance, and Seasonal Activity of Sphenophorus spp. in Florida...... 17 Sphenophorus venatus vestitus Adult Activity Patterns, Fecundity and Generation Time ...... 18 Adult daily activity patterns ...... 18 Potential fecundity and egg development of S. venatus vestitus...... 19 Variation of adult S. venatus vestitus body size ...... 20 Length of S. venatus vestitus Development in Bermudagrass and Zoysiagrass...... 20 Larval instar determination ...... 21 Results...... 21 Composition, Abundance, and Seasonal Activity of Sphenophorus spp. in Florida...... 21 Sphenophorus venatus vestitus Adult Activity Patterns, Fecundity and Generation Time ...... 23 Adult daily activity patterns ...... 23 Potential fecundity and egg development of S. venatus vestitus...... 23 Variation of adult S. venatus vestitus body size ...... 24 Length of S. venatus vestitus development in bermudagrass and zoysiagrass...... 24 Larval instar determination ...... 25 Discussion...... 25
3 EVALUATION OF BEMUDAGRASS AND ZOYSIAGRASS RESISTANCE TO Sphenophorus venatus vestitus ...... 42
Materials and Methods ...... 43 Damage potential of adult S. venatus vestitus on Fur Brmudagrass Cltivars...... 43
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Plant material...... 43 Adult damage potential ...... 43 Bermudagrass Cltivar Peference Test ...... 44 Plant material...... 44 Preference evaluation ...... 44 Zoysiagrass Cultivar Resistance to S. venatus vestitus ...... 44 Plant material...... 44 Resistance evaluation ...... 45 Results and Discussion ...... 46 Damage Potential of Adult S. venatus vestitus on Four Bermudagrass Cultivars...... 46 Bermudagrass Cultivar Preference Test ...... 46 Zoysiagrass Cultivar Resistance to S. venatus vestitus ...... 47
4 EFFECT OF ENDOPHYTE LEVEL IN PERENNIAL RYEGRASS ON THE SURVIVAL AND DEVELOPMENT OF Sphenophorus venatus vestitus...... 56
Materials and Methods ...... 57 Sphenophorus venatus vestitus Survival, Development, and Damage on Four Endoyphytic Perennial Ryegrass Cultivars...... 57 Impact of Overseeding Two Bermudagrass Cultivars with Endophytic Perennial Ryegrass on S. venatus vestitus Survival, Development, and Damage...... 57 Overseeded Bermudagrass Field Trial ...... 58 Results and Discussion ...... 59 Sphenophorus venatus vestitus Survival, Development, and Damage on Four Endoyphytic Perennial Ryegrass Cultivars...... 59 Impact of Overseeding Two Bermudagrass Cultivars with Endophytic Perennial Ryegrass on S. venatus vestitus Survival, Development, and Damage ...... 59 Overseeded Bermudagrass Field Trial ...... 60
APPENDIX
A EVALUATION OF BEMUDAGRASS RESISTANCE TO Sphenophorus inaequalis...... 63
Materials and Methods ...... 63 Damage Potential of Adult S. inaequalis on Four Bermudagrass Cultivars ...... 63 Plant material...... 63 Adult damage potential ...... 63 Results and Discussion ...... 64 Damage Potential of Adult S. inaequalis on Four Bermudagrass Cultivars ...... 64
LIST OF REFERENCES...... 66
BIOGRAPHICAL SKETCH ...... 71
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LIST OF TABLES Table Page
2-1 Total number of Sphenophorus spp. collected from four linear pitfall traps at each of four Florida golf courses during 24-hour sample periods from January 2006 to December 2007...... 30
2-2 Mean (±SEM) number of eggs per female S. venatus vestitus collected from three golf courses in Florida in 2007...... 31
2-3 Mean (±SEM) number of eggs per female S. inaequalis collected at West End Country Club in 2007...... 32
2-4 Mean (± SEM) body lengths of male and female S. venatus vestitus at three golf courses in 2007...... 33
3-1 Damage between male and female S. venatus vestitus on four bermudagrass cultivars in the greenhouse...... 50
3-2 Mean (±SEM) number of notches and surviving larvae per pot on four bermudagrass cultivars in the greenhouse preference test...... 51
3-3 Ratings (±SEM) of seventeen zoysiagrass cultivars after 4 weeks of adult S. venatus vestitus infestation...... 52
3-4 Mean number (±SEM) of S. venatus vestitus eggs and larvae, and characteristics of adult notches on 17 zoysiagrass cultivars, after 1 month of adult confinement...... 53
3-5 Mean number (±SEM) of S. venatus vestitus eggs and larvae, and adult notches on 18 zoysiagrass cultivars, after one month of adult confinement...... 54
4-1 The effect of overseeding with endophytic perennial ryegrass on two bermudagrass cultivars on S. venatus vestitus damage potential and oviposition ...... 62
A-1 Damage between male and female S. inaequalis on four bermudagrass cultivars in the greenhouse ...... 65
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LIST OF FIGURES
Figure Page
2-1 Total number of S. venatus vestitus adults collected weekly from four linear pitfall traps at Gainesville Country Club...... 344
2-2 Total number of adult S. venatus vestitus obtained weekly from four linear pitfall traps at LaGorce Country Club...... 355
2-3 Total number of adult S. venatus vestitus trapped weekly at Card Sound Country Club...... 366
2-4 Total number of S. inaequalis adults collected each week from four linear pitfall traps at West End Country Club...... 377
2-5 Mean number of S. venatus vestitus adults that were active on the soil surface of bermudagrass pots during a 24-hour period of observation...... 389
2-6 Number of life stage for S. venatus vestitus reared on pots of ‘Tifway’ bermudagrass in the greenhouse...... 39
2-7 Number of life stage for S. venatus vestitus reared on pots of ‘Empire’ zoysiagrass in the greenhouse...... 40
3-1 Sphenophorus venatus vestitus preference test for bermudagrass cultivars...... 555
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Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science
BILLBUG (Sphenophorus spp.) COMPOSITION, ABUNDANCE, SEASONAL ACTIVITY, DEVELOPMENT TIME, CULTIVAR PREFERENCE, AND RESPONSE TO ENDOPHYTIC RYEGRASS IN FLORIDA
By
Ta-I Huang
May 2008
Chair: Eileen A. Buss Major: Entomology and Nematology
Billbugs (Sphenophorus spp.) are common pests whose damage is often misdiagnosed on turfgrass in the United States. Florida turfgrass managers experiencing billbug outbreaks have been struggling to obtain satisfactory control using conventional insecticide applications. Thus, I have sought to identify the billbug (Sphenophorus spp.) complex, seasonal activity, and development time in golf course bermudagrass, document billbug responses on bermudagrass and zoysiagrass cultivars, and determine the effect of endophytic perennial ryegrass on billbug development.
Adult billbug populations were monitored weekly from January 2006 to December 2007 with four linear pitfall traps on each of two bermudagrass golf courses in southern Florida and two in north-central Florida. Several studies of bermudagrass and zoysiagrass cultivar resistance were conducted in the greenhouse by caging adult Sphenophorus venatus vestitus onto plastic pots in 2006 and 2007. The total number of notches, eggs, and larvae were recorded to evaluate resistance. Overseeding of endophytic perennial ryegrass into bermudagrass was conducted in the greenhouse and in the field to evaluate the impact to S. venatus vestitus oviposition and larval
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development. Four endoyphytic perennial ryegrass cultivars were also tested for S. venatus
vestitus feeding damage and oviposition potential.
My results suggested that S. venatus vestitus is the most abundant billbug species on
Florida golf courses containing bermudagrass, constituting >80% of the specimens collected from the 24-hr samples, and the remainder consists of nine other Sphenophorus spp. As many as
686 individuals of S. venatus vestitus were collected within a 24-hr sampling period during peak
adult activity in April 2006 on one golf course in southern Florida, but activity was steady on the other three courses (<150 adult billbugs per 24-hr sample). Sphenophorus venatus vestitus shows
greater preference for ‘Tifway’ bermudagrass than other cultivars, and Zoysia matrella cultivars
display higher resistance than cultivars of Zoysia japonica. Bermudagrass overseeded with
endophytic perennial ryegrass significantly reduced S. venatus vestitus feeding damage and
oviposition.
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CHAPTER 1 INTRODUCTION
Warm Season Grasses in North America
Warm-season turfgrasses are widely grown in the transition zone and southern part of the
United States. These typical warm season turfgrasses include bahiagrass (Paspalum notatum
Fluegge), bermudagrass (Cynodon dactylon (L.) Pers), centipedegrass (Eremochloa ophiuroides (Munro) Hack.), seashore paspalum (Paspalum vaginatum Sw.), St. Augustinegrass
(Stenotaphrum secundatum (Walt.) Kuntze.), and zoysiagrass (Zoysia spp.). Bermudagrasses are among the most widely used warm-season grasses on golf courses, athletic fields, and in high- profile residential and commercial landscapes where a fine-textured, dense ground cover is desired (Trenholm 2003). ‘Tifway’, ‘Tifdwarf’, and ‘Tifeagle’ are the most popular bermudagrass cultivars used on golf course roughs, fairways, and greens in Florida (Russ Meyer, personal communication 2006). With over 1,500 golf courses, Florida turfgrass managers and golfers demand high quality turf. However, turfgrass quality can be impaired by insects that feed on grass leaves, stems, sap, or roots (Buss 2003).
Common Arthropod Pests of Bermudagrass
Common arthropod pests that feed on bermudagrass include mole crickets (Scapteriscus spp.), several caterpillar species, bermudagrass mites [Eriophyes cynodoniensis (Sayed)], white grubs (Coleoptera: Scarabaeidae), and billbugs (Sphenophorus spp.). Mole crickets damage turfgrass by tunneling in the soil, uprooting plants, and feeding on turfgrass roots (Buss et al.
2006). Several species of caterpillars [e.g., Spodoptera spp., Mocis spp.] skeletonize and completely consume grass leaf blades, causing an uneven and off-colored appearance to the turfgrass (Buss and Turner 2004). Feeding by the bermudagrass mite results in shortened stems and stolons, yellow and curly blades, and tufts of grass plants (Short and Buss 2005). White
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grubs (especially masked chafers, Cyclocephala spp.) feed on fine turfgrass roots, which makes bermudagrass turn slightly yellow, but they also are food for birds that dig in the turf to find them (Buss and Turner 2004). Billbug larvae are stem borers and root feeders, and adults can damage stems, but their damage is often misdiagnosed as drought stress or disease pressure.
Infested grass turns yellow and results in patchy spots during a heavy infestation (Brandenburg and Villani 1995).
Billbugs Sphenophorus spp. in the United States
Although over 60 native billbug species exist, roughly nine species have been reported to damage turfgrass in North America: the bluegrass billbug, S. parvulus Gyllenhal; the lesser billbug, S. minimus Hart; S. venatus Say (no common name); the uneven billbug, S. inaequalis
Say; the hunting billbug, S. venatus vestitus Say; the Phoenician billbug, S. phoeniciensis
Chittenden; the Denver billbug, S. cicatristriatus Fabraeus; S. coesifrons Gyllenhal; and S. apicalis LeConte (Vaurie 1951, Tashiro 1987, Morrill and Suber 1976, Johnson-Cicalese et al.
1990).
Sphenophorus parvulus is the most common pest of cool-season turf, and attacks Kentucky bluegrass, perennial ryegrass, fine fescue and tall fescue from the Pacific Northwest across to
New England (Tashiro 1987). It has also been reported in several of the Gulf States where warm- season turfgrasses predominate (Richmond 2000). Sphenophorus minimus occurs in the northeastern U.S., feeding on cool-season turfgrasses. The life cycle and host range of S. minimus is very similar to S. parvulus (Johnson-Cicalese et al. 1990, Shetlar 1991). The distribution of S. inaequalis is limited to the eastern United States (Veurie 1951). It attacks both warm and cool season turfgrasses including bermudagrass, Kentucky bluegrass, perennial ryegrass, and tall fescue. The life cycle of S. inaequalis is similar to S. parvulus and its population could be as abundant as S. parvulus in New Jersey (Johnson-Cicalese and Funk
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1990). The most common billbug in warm season turfgrass is S. venatus vestitus. It occurs north
to New Jersey and west to Texas, and is most abundant in and damaging to bermudagrass and
zoysiagrass. Adult females chew holes or notches in the grass stems where they deposit their
eggs (Brandenburg and Villani 1995). The distribution of S. phoeniciensis is limited to southern
California and Arizona. Its life cycle and hosts are similar to S. venatus vestitus (Tashiro 1999).
Sphenophorus cicatristriatus occurs in the Rocky Mountain region from northern New Mexico
to Nebraska and prefers feeding on cool season turfgrasses such as Kentucky bluegrass and
perennial ryegrass (Vaurie 1951, Tashiro 1987, Shetlar 1995). Sphenophorus cicatristriatus
overwinters as a late instar larva, and the adult population peaks between June and mid-
September (Vaurie 1951, Shetlar 1995). The life cycle of both S. coesifrons and S. apicalis are
poorly understood. The distribution of S. coesifrons extends throughout the southern United
States while S. apicalis occurs from New Jersey to the Gulf States (Vaurie 1951).
Sphenophorus venatus vestitus in the Southeastern United States
The species considered most abundant and damaging in the southeastern United States is S.
venatus vestitus. Adults (6-11 mm long) cause notches while feeding on stem tissue. Legless
larvae are usually white to yellowish with brown head capsules. First instar larvae feed inside stems and then drop out into the soil. Mature larvae feed on roots in soil and pupate in a chamber
about 2 to 5 cm deep (Brandenburg and Villani 1995). Sphenophorus venatus vestitus occurs
from New Jersey to Florida and along the Gulf states, west to Texas. It is also present in Mexico
and some of the Caribbean islands, and was transported with sod to the Middle East, Southeast
Asia and Hawaii (Vaurie 1951, Tashiro 1999, Niemczyk and Shetlar 2000).
The most commonly recorded hosts in Florida are bermudagrass and zoysiagrass. Other
minor hosts include bahiagrass, centipedegrass, St. Augustinegrass, orchardgrass (Dactylus
glomeratus L.), crabgrass (Digitaria spp.), signal grass (Brachiaria decumbens Stapf),
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barnyardgrass (Echinochloa crusqalli Beauv.), corn (Zea mays L.), sugarcane (Saccharum officinarum L.), and leather leaf fern (Polypodium scouleri Hook and Grev.) (Satterthwait 1919,
Kelsheimer 1956, Kamm 1969, Oliver 1984). Satterthwait (1931) listed some additional hosts of
S. venatus vestitus: yellow nutsedge (Cyperus esculentus L.), wheat (Triticum aestivum L.),
timothy (Phleum pratense L.), and a bulrush (Scirpus validus Vahl.) (Woodruff 2005).
Billbug Integrated Pest Management
Billbugs are considered one of the most misdiagnosed pests of turfgrass (Potter 1998). Turf
managers usually misdiagnose billbug damage symptoms as drought stress, delayed green up in
the spring, disease such as dollar spot, or other insect feeding damage (Buss 2006). Billbug
infestations may be managed using resistant turfgrasses, overseeding with endophyte-enhanced
turfgrasses, biopesticides and preventive or curative insecticides. Asay et al. (1983) reported a
genetic resistance of range grasses to S. parvulus. Later, several studies demonstrated endophyte-
enhanced resistance in perennial ryegrass to S. parvulus and other Sphenophorus spp. (Ahmad et
al. 1986, Jonhson-Cicalese and White 1990, and Richmond 2000).
Little is known about the biological control of billbugs, but their natural enemies include
parasitoids, nematodes, and fungi. Satterthwait (1919) reported a parasitic wasp, Vipio belfragei
Cresson, reared from billbug larvae, and a mymarid egg parasitoid, Anaphoidea calendrae
Gahan, was documented to parasitize several Sphenophorus spp. (Satterthwait 1931). Several
entomopathogenic nematodes (e.g., Steinernema carpocapsae, S. feltae, and Heterorhabditis
bacteriophora) suppress both white grubs and billbugs (Niemczyk and Shetlar 2000).
Muscardine fungi, Beauveria spp., were found to attack several billbug species in New Jersey
(Johnson-Cicalese 1988), but their effectiveness is unknown.
A comprehensive knowledge of the billbug life cycle and seasonal phenology is critical for insecticidal control. A preventive application in spring for billbug control is recommended by
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most extension reports and turfgrass management websites. The goal is to kill adults emerging in the spring before they lay eggs, or kill first instars as they emerge and begin feeding. Early season applications to manage S. parvulus have been effective in western New York lawns
(Tashiro and Personius 1970). However, regions with multiple, overlapping generations and a prolonged adult activity period may have difficulty controlling infestations only with insecticides.
An integrated pest management (IPM) program needs to be developed for billbugs on warm- season turfgrasses.
Given the limited information about general billbug biology and management in warm season turfgrasses in Florida, I sought to describe the species composition, abundance, seasonal activity, developmental time of Sphenophorus spp. on golf courses utilizing bermudagrass
(Chapter 2), document billbug preference on bermudagrass and zoysiagrass cultivars (Chapter 3), and billbug response to endophytic perennial ryegrass (Chapter 4).
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CHAPTER 2 BIOLOGY AND SEASONAL PHENOLOGY OF BILLBUGS, Sphenophorus spp., IN FLORIDA
Over 60 native billbug species exist in the United States (Niemczyk and Shetlar 2000), and at least nine of those species attack turfgrass (Vaurie 1951, Morrill and Suber 1976,
Johnson-Cicalese et al. 1990). The bluegrass billbug, Sphenophorus parvulus Gyllenhal, typically infests cool season turfgrasses in the northeastern United States (Tashiro 1987), including Kentucky bluegrass (Poa pratensis L.), perennial ryegrass (Lolium perenne L.), tall fescue (Festuca arundinacea Schreb.), and 14 other non-turf grasses (Johnson-Cicalese and
Frank 1990). The lesser billbug, S. minimus Hart, also infests cool season grasses in the northeastern United States (Johnson-Cicalese et al. 1990, Shetlar 1991). The uneven billbug, S. inequalis Say, occurs in the eastern United States on Kentucky bluegrass, perennial ryegrass, tall fescue and bermudagrass (Cynodon dactylon (L.) Pers.) (Satterthwait 1931, Johnson-Cicalese and Frank 1990). Little is known about S. apicalis LeConte, but it occurs in the Gulf States and
New Jersey (Vaurie 1951). Sphenophorus coesifrons (no common name) is distributed throughout the southern United States (Vaurie 1951), and attacks bahiagrass (Paspalum notatum
Flüggé.) (Morrill and Suber 1976). The Denver billbug, S. cicatristriatus Fahraeus, occurs in the
Rocky Mountain region from northern New Mexico to Nebraska and prefers feeding on cool season turfgrasses such as Kentucky bluegrass and perennial ryegrass (Vaurie 1951, Tashiro
1987, Shetlar 1995). The Phoenician billbug, S. phoeniciensis (Chittenden), infests warm season grasses in southern California and Arizona, and the orchardgrass billbug, S. venatus confluens
Chittenden, is found in Oregon. Its primary host is orchardgrass (Dactylus glomeratus L.)
(Kamm 1969). The hunting billbug, S. venatus vestitus Chittenden, infests primarily bermudagrass and zoysiagrass (Zoysia spp.) from New Jersey to Florida and along the Gulf
States (Tashiro 1987).
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Billbugs are both stem boring and root feeding turfgrass insects (Potter 1998). Adult females chew holes or notches into grass stems that are utilized for egg deposition (Brandenburg and Villani 1995). Eggs (about 1.5 mm long) are clear to creamy white, and hatch in 3-10 days
depending on the temperature (Potter 1998). Young larvae (about 1.3 mm long) tunnel in and
hollow out stems, and leave sawdust-like frass inside the stems (Potter 1998). Mature larvae
(about 6-10 mm long) severely damage grass by feeding on crowns, stolons, and roots. Pupae
(about 1.3-1.5 mm long) occur in a soil chamber 2.5-5.1 cm deep (Shetlar 1995). Adults (8-11
mm in length) have elongate snouts, elbowed antennae, and hard elytra (Brandenburg and Villani
1995), and different species may be identified by unique pronotal patterns (Vaurie 1951,
Johnson-Cicalese et al. 1990).
Although 25 species of billbugs occur in Florida (Peck and Thomas 1998), only S. venatus
vestitus is credited as being the most abundant and damaging species. The seasonal activity of S.
venatus vestitus is poorly known in Florida, and although it has only one generation per year in
northern states, it has been speculated that several overlapping generations occur along the Gulf
States (Potter 1998). My goals here include 1) Determining which Sphenophorus spp. are most
abundant in northern and southern Florida 2) Determining the development time and seasonal
phenology of S. venatus vestitus and S. inaequalis.
Materials and Methods
Composition, Abundance, and Seasonal Activity of Sphenophorus spp. in Florida
The composition, abundance, and seasonal activity of Sphenophorus spp. on four golf
courses in Florida were determined weekly by taking 24-hour samples with large linear pitfall
traps from January 2006 to December 2007. The golf courses included Gainesville Country Club
and West End Country Club in Gainesville, LaGorce Country Club in Miami Beach, and Card
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Sound Country Club in Key Largo, Florida. Four linear pitfall traps (similar to Lawrence 1982) were placed in bermudagrass (Cynodon dactylon (L.) Pers.) roughs on each golf course in
January 2006. Each trap had four 3-m long “arms” of PVC pipe (7.6 cm diameter), with a 2.5 cm slit cut lengthwise in each arm. The far pipe end was capped, and the inner pipe end was placed in a hole on the side of a 19 liter bucket. A removable plastic tube extended the arm over a removable 4 liter bucket. Rocks were placed beneath the 19 liter bucket, and both buckets had
holes drilled in the bottom to allow water drainage. To sample, debris was removed from traps in
the morning, sand was added to the bottom of the 4 liter bucket, and any billbugs caught during
the following 24-hours were collected and preserved in 70% EtOH. Adult billbug species,
abundance and gender were determined for each sampling date. Weather conditions for each
sampling period were recorded.
The soil activity of Sphenophorus spp. was examined at the same golf courses monthly from 24 January 2006 to 15 December 2006. Four cores (10.2 cm diameter, 15 cm deep) were collected near damaged areas on bermudagrass roughs. Cores were placed in plastic bags,
transported to the laboratory, and dissected under at least 10X magnification. Any billbug life
stages present were collected, counted, and preserved in 70% EtOH. This sampling method was
discontinued in 2007 because few specimens were recovered.
Sphenophorus venatus vestitus Adult Activity Patterns, Fecundity and Generation Time
Adult daily activity patterns
The diurnal activity patterns of adult S. venatus vestitus were observed every 4 hours each
day on 30 April 2007 (15 pots), 11 November 2007 (10 pots), 15 January 2008 (10 pots), and 7
February 2008 (10 pots) (7 observation intervals). Two male and two female S. venatus vestitus
adults were placed onto each ‘Tifway’ bermudagrass pot (11.4 cm diameter). Adults were
allowed to acclimate for 2 days before observations began. The adults in each pot were observed
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for 20 seconds, and the location of adults (e.g., grass blade, soil surface, not visible) and their
behavior (e.g., grooming, walking, feeding, mating, or inactive) were recorded (but not timed).
Red light was used for night observations to minimize disturbance. Weather conditions were
recorded at each observation period. Data were analyzed using two-way ANOVAs (SAS
Institute 2000) to detect the effect of time and date on billbug activity. Means were compared
using Tukey’s HSD test (P < 0.05).
Potential fecundity and egg development of S. venatus vestitus
Adult female S. venatus vestitus collected from the linear pitfall traps at the four golf courses from 22 January to 27 December 2007 were dissected with a 10X dissecting microscope.
Twenty or more females were dissected at least monthly from each site. The number of mature
eggs (1.5 to 1.7 mm long) in their abdomens was recorded. The time of ovarial development at
each golf course, and whether or not females were in reproductive diapause were determined by
examination of the ovaries (Young 2002). Data were analyzed using two-way ANOVAs (SAS
Institute 2000) to detect the effect of month and site on female S. venatus vestitus potential
fecundity. Means were compared using Tukey’s HSD test (P < 0.05).
Twenty female S. venatus vestitus were collected from pitfall traps at Gainesville Country
Club on 9 August 2006 and placed in a container (15 × 15 cm) with moistened filter paper.
Females readily laid eggs in the absence of plant tissue. Twenty-five newly laid eggs (<24 hrs
old) were collected from the container and placed in a Petri dish (8.8 cm diameter) with moistened filter paper in a rearing room under 24-26°C, 25% relative humidity, light intensity of
~4.05 lum/m2, and a photoperiod of 10:14 (L:D) hrs. The percentage of eggs hatching each day was determined until all eggs had hatched or died.
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Variation of adult S. venatus vestitus body size
Twenty male and 20 female adult S. venatus vestitus that were collected from the pitfall
traps at Gainesville Golf and Country Club, LaGorce Country Club, and Card Sound Country
Club each in March, July, and November 2006 were examined for differences in body size. The
body length from the base of the beak to the tip of the anus was measured with a 10X dissecting
microscope and ocular micrometer. Data were analyzed using a two-way ANOVA (SAS Institute
2000) to detect the effect of time and site on S. venatus vestitus body length. Means were
compared using Tukey’s HSD test (P < 0.05).
Length of S. venatus vestitus Development in Bermudagrass and Zoysiagrass
Thirty plastic pots (11.4 cm diameter) were planted with cores of Tifway bermudagrass on
30 March 2007, and 15 pots each of ‘Empire’ and ‘Pristine’ zoysiagrass were similarly potted on
4 May 2007, and allowed to establish in the greenhouse for one month. All cores were obtained from the University of Florida’s Plant Science Research Unit in Citra, FL. Pots were fertilized
with 113.5 g of Miracle-Gro® all purpose fertilizer (20-20-20) per week and irrigated daily.
Sixty pairs of adult S. venatus vestitus were collected on Tifway bermudagrass on 27 April 2007
and on Empire zoysiagrass on 19 June 2007 at the U.F. Plant Science Research Unit in Citra, FL.
Two pairs of S. venatus vestitus were randomly chosen and caged onto each pot within 24 hours
of collection. Each pot was encircled up to 15 cm above the turf height with a clear plastic tube
to prevent adult escape. All adults were removed from pots five days after introduction. Six
different pots were randomly selected and destructively sampled to find all billbug life stages at
3, 6, 9, 11, and 12 weeks post-adult removal in the bermudagrass trial and every 2 weeks in the
zoysiagrass trial (2, 4, 6, 8, 10 weeks post-adult removal). Larvae were preserved in KAAD for
48 hours and stored in 70% EtOH. Air temperature, relative humidity, and soil temperature were
recorded in the greenhouse throughout the experiment.
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Larval instar determination
To estimate the number of instars for S. venatus vestitus, the head capsule widths of 135
larvae reared on bermudagrass were measured under a 10X dissecting microscope equipped with
an ocular micrometer. First instar head capsule widths were measured on larvae that hatched
from eggs that females laid in a Petri dish (8.8 cm diameter) with moistened filter paper.
Results
Composition, Abundance, and Seasonal Activity of Sphenophorus spp. in Florida
From January 2006 to December 2007, a total of 18,580 adult billbugs of ten different
Sphenophorus spp. were collected from the four golf courses (Table 2-1). Sphenophorus venatus vestitus was the most abundant species collected, constituting 80.9% of all specimens (Table 2-
1). Gainesville Golf and Country Club and West End Country Club had the greatest species diversity, each with eight species collected during the 24-hour sampling periods. LaGorce
Country Club had the lowest species diversity, with three species collected. Sphenophorus
venatus vestitus was the primary species (99.9% of all specimens) collected from the two
southern Florida golf courses. The sex ratio of all the samples combined was 1.1:1 (males to
females) for S. venatus vestitus, 1.9:1 for S. inaequalis, and 1.2:1 for S. cariosus Olivier. Billbug
gender was determined by examining the ventral side of the abdomen; males had a full, rounded
abdomen, and female abdomens were depressed. In addition to the specimens collected during
the 24-hour sampling period, six additional Sphenophorus spp. were found when traps were
cleaned, including S. callosus Olivier, S. coesifrons Gyllenhal, S. minimus Hart, S. necydaloides
Fabricius, S. pontederiae Chittenden, and S. dietrich Satterthwait (a new Alachua County record).
Sphenophorus inaequalis is a new Monroe County record and S. costipennis Horn is a new
Florida state record (Peck and Thomas 1998).
21
Adult S. venatus vestitus were collected from pitfall traps nearly every week of the year.
The timing of peak trap catches of S. venatus vestitus varied on each golf course. At Gainesville
Golf and Country Club, it was most abundant on 7 March 2006 (68 adults per 4 traps), at
LaGorce Country Club on 10 April 2006 (685 adults), and at Card Sound Country Club on 4 July
2007 (163 adults) (Figures 2-1 to 2-3). The months of greatest overall S. venatus vestitus adult activity were March and April. Specimens were not collected on several dates because traps were flooded or cooperators at the two southern golf courses were unable to check them. Given the variability of adult S. venatus vestitus activity over time and location, the number of generations per year could not be determined through pitfall trapping.
The second most abundant species collected was S. inaequalis, constituting 17.8% of all
specimens (Table 2-1). It was collected at three of the four golf courses, but was most abundant
at West End Country Club. Peak abundance of S. inaequalis at West End Country Club was on
17 July 2006 with 145 adults collected during the 24 hour sampling period. The mean number of
S. inaequalis adults collected by month was highest in July 2006 (108.5 ± 12.7) and lowest in
February 2006 (13.2 ± 7.2) (Figure 2-4).
No eggs or young larvae were found from turf in the cupcutter samples collected in 2006.
Nearly mature larvae were only found in the soil. With the results from all four golf courses
combined, four larvae were collected in March, three in April, one in May, four in July, and one
in October 2006. One pupa was recovered in March. One adult was collected in April, one in
May, two in June, one in July, one in September, and two in October. Species identification of
immature Sphenophorus spp. was not possible.
22
Sphenophorus venatus vestitus Adult Activity Patterns, Fecundity and Generation Time
Adult daily activity patterns
Adults were seen feeding, mating, walking, or inactive at all observation times, but were
most active on the soil surface between midnight and 4:00 am (F = 52.79; df = 5, 314; P <
0.0001). The mean number of adults observed on each date was also significantly different (F =
26.82; df = 3, 314; P < 0.0001). Mating and grooming were only observed between midnight and
4:00 am (20% and 5% of adults, respectively). During observations air temperature ranged from
18.6 - 29.5 °C, soil temperature ranged from 63 - 72°C, and relative humidity ranged from 40 -
86%. The mean number of adults observed on the surface between midnight and 4:00 am on 1
May 2007 (F = 13.18; df = 6, 104; P < 0.0001), 11 November 2007 (F = 8.95; df = 6, 69; P <
0.0001), 15 January 2008 (F = 5.21; df = 6, 69; P = 0.0005), and 7 February 2008 (F = 20.05; df
= 6, 69; P < 0.0001) were all significantly higher than any other time period (Figure 2-5). The
interaction between each observation time and date was not significantly different.
Potential fecundity and egg development of S. venatus vestitus
The potential fecundity of S. venatus vestitus varied by location and time of year. Mature
eggs were found from female ovaries almost every month of the year on each golf course except
for June at LaGorce Country Club and November at Card Sound Country Club. Number of
mature eggs per female were 0 to 14 (4.9 ± 0.1) at the Gainesville Golf and Country Club, zero
to 14 (4.9 ± 0.1), 0 to 9 (3.1 ± 0.2) at LaGorce Country Club, and 0 to 9 eggs (3.4 ± 0.16) at Card
Sound Country Club (Table 2-2). From all data combined, female S. venatus vestitus collected
from Gainesville Golf and Country Club contained significantly more mature eggs than both golf
courses in southern Florida (F = 13.5; df = 2, 33; P < 0.0001). Fecundity was greatest in March
at LaGorce Country Club (F = 5.03; df = 10, 233; P < 0.0001) and in October at Gainesville Golf
and Country Club (F = 2.88; df = 11, 654; P = 0.0011). From the Petri dish rearing, 44 % (11
23
eggs) of the eggs hatched 7 days after oviposition and 40% (10 eggs) after 8 days. The remaining
four eggs died within 6 days of oviposition.
At West End Country Club, the number of mature eggs found in female S. inaequalis
ovaries ranged from 0 to 5 (1.9 ± 0.05) (Table 2-3). Sphenophorus inaequalis females contained
significantly more mature eggs during the months of March, July and September than other
months (F = 6.52; df = 11, 669; P < 0.0001).
Variation of adult S. venatus vestitus body size
The average body length of female S. venatus vestitus (8.3 ± 0.03 mm; range: 7.1 to 9.6
mm) was significantly greater than for males (7.5 ± 0.03 mm; range: 6.6 to 8.4 mm) (F = 320.69; df = 1, 359; P < 0.0001), regardless of time or location. Body lengths of males and females collected from Gainesville Golf and Country Club were significantly greater than for males and females collected from LaGorce Country Club and Card Sound Country Club (male: F = 8.05; df
= 2, 179; P = 0.0005) (female: F = 15.51; df = 2, 179; P < 0.0001) (Table 2-4). Body lengths of all S. venatus vestitus collected in March, July, and November combined, did not significantly differ among golf courses.
Length of S. venatus vestitus development in bermudagrass and zoysiagrass
Bermudagrass. From 24 May to 26 July 2007, a total of two eggs, 45 larvae, seven pupae,
and 17 adults were found during the five evaluations. At the first time evaluation, 24 May 2007,
only two eggs and ten larvae were found (two larvae were in the soil and the rest were in the
stems). Thirty-one larvae and only one pupa were found on 14 June 2007. Three larvae, three
pupae and two adults were found on 5 July 2007. One larva, three pupae, and seven adults were
found on 19 July 2007. Eight adults were found on 26 July 2007 (Figure 2-6).
Zoysiagrass. From 7 July to 3 September 2007, a total of five eggs, 24 larvae, two pupae,
and eight adults were found in the five evaluations. Five eggs and seven larvae were found on 9
24
July 2007. Five larvae were found on 23 July 2007. Seven larvae and one pupa were found on 6
August 2007. Five larvae, one pupa, and one adult were found on 20 August 2007. Seven adults
were found on 3 September 2007 (Figure 2-7).
Larval instar determination
The head capsule width of S. venatus vestitus larvae ranged from 0.4 to 2.4 mm. The head
capsule width of first instars was about 0.5 ± 0.02 mm (n = 20), and the head capsule width of
late instar larvae was about 2.3 ± 0.02 mm (n = 15). The number of instars could not be
determined, but 4-5 instars may exist, based on the frequency distribution (Figure 2-8).
Discussion
Despite the potential for a large species complex, S. venatus vestitus was the most
abundant species present on bermudagrass in the four golf courses monitored. It was also expected to be the most damaging species, given its reputation in warm season turfgrass (Tashiro
1987), but distinct damage was only visible on LaGorce Country Club, which had the highest
number of adult S. venatus vestitus collected throughout the study (Table 2-1).
The composition of the billbug species complex varied between northern and southern
Florida and by golf course. Species composition may vary in the state due to different
horticultural zones, soil types, or management practices, but Tifway bermudagrass was
commonly grown at all four locations and bermudagrass is widespread on golf courses
throughout the state. Sphenophorus venatus vestitus has a broad geographic range in North
America, spanning both cool and warm season grasses (Johnson-Cicalese et al. 1990). Young
(2002) also reported similar numbers of S. venatus vestitus in Arkansas. However, Johnson-
Cicalese et al. (1990) reported that the four billbug species (S. inaequalis, S. minimus, S.
parvulus, and S. venatus vestitus) occurred nearly equal numbers in New Jersey. In my study, S.
25
minimus and S. parvulus were only found in Gainesville and their populations were smaller
compared to S. venatus vestitus.
The life cycle of S. venatus vestitus and body size appears to also vary with location, but
may be more greatly influenced by temperature or host plant. In general, S. venatus vestitus overwinters as adults in northern states, adult population increases in spring, then declines in summer, another smaller population increases in the fall and decreases in winter (Johnson-
Cicalese et al. 1990, Young 2002). It has one generation per year in northern states (Tashiro
1987), and may have a partial second generation in New Jersey (Johnson-Cicalese et al. 1990). It
is also suspected to have two generations in Georgia (S. K. Braman, personal communication).
The development from oviposition to adult emergence in my greenhouse study at ca. 25.8 °C
took ca. 10-11 weeks on bermudagrass and ca. 27 °C took ca. 8-10 weeks. In addition, female S.
venatus vestitus develop mature eggs every month of the year and can live at least one month.
Therefore, it is possible that S. venatus vestitus has at least 2 to 3 overlapping generations each
year in Florida. Another similar species, S. venatus confluens, overwinters as adults, begins to
feed in March, and adults die in July as larvae emerge and feed throughout the summer (Kamm
1969). However, the seasonal activity from my pitfall trapping data in Florida showed different
adult activity by different locations. Sphenophorus venatus vestitus adults were most abundant in
March and April. The population then decreased during the summer followed by a smaller peak
again during late fall. Difference in climate may affect in the activity patterns between southern
and northern Florida. The pitfall trap data are difficult to interpret because of the variability in
infestation levels among the golf courses sampled.
Adult activity pattern of Sphenophorus spp. within a 24-hour period is fairly unknown. An
observation study on the southern corn billbug, S. callosus, indicated that adults were most active
26
during daylight hours (DuRant 1985). Activity was more associated with air temperature than
with soil temperature, and was inhibited when air temperature was below 20oC (DuRant 1985).
However, adult observations in this study showed an opposite activity period from the previous
study. Adult S. venatus vestitus activity was greater at midnight than any other timing, which suggests that S. venatus vestitus adults may be nocturnal. Although some behavior was observed during the day, most adults emerged on the soil surface to feed and mate at night. However, billbug activity may last until early morning since many of Sphenophorus spp. are observed on sidewalk in early morning (O’Brien, 2006 personal communication).
Johnson-Cicalese et al. (1990) reported a long oviposition period of S. venatus vestitus from spring to early September in New Jersey. In Arkansas, ovarian development of S. venatus vestitus was quickly mature in early April, and remained highly mature until October (Young
2002). In Oregon, S. venatus confluens deposit a large number of eggs in the stems and on the leaf sheaths in mid-June (Kamm 1969). In this study, the number of eggs deposited in grass could not be determined from golf courses, so the precise time of ovarial maturation was determined by dissecting females from weekly trap collections. In general, mature eggs of S. venatus vestitus were found every month in 2007, but were higher in early spring and late fall than in summer. Mature eggs were still presented in December which may suggest a second generation occurred in Florida, and S. venatus vestitus overwinters in both adult and immature stages.
The reason why the fecundity of S. venatus vestitus in Gainesville was significantly higher than in southern Florida is unknown. One possible explanation may be the difference in body length. Since a lot of species of Coleoptera have been proved than female size is a principal constraint on insect potential fecundity (Honek 1993), the significantly longer body length of S.
27
venatus vestitus in Gainesville (Table 2-4) resulting in higher fecundity than in south Florida is reasonable. The other possibility may be population density. Reigada and Godoy (2005) reported that fecundity and body size are generally density-dependent characters influenced by insect populations. Female S. venatus vestitus in Gainesville have to produce more eggs because of its low population and greater species diversity; In contrast, female S. venatus vestitus in south
Florida may not need high fecundity since the population is large and there is less species competition (Table 2-1).
In greenhouse rearing studies, adult billbugs were caged into plastic pots by encircling with clear plastic tubes 15 cm above the turf height. This method proved excellent for preventing adults from escaping. Since flying activity was only observed in a container twice for S. venatus vestitus, and several times for S. inaequalis. I speculate that crawling is the major method of
population dispersal. Young (2002) reported that flight occurs sporadically by only a few S.
venatus vestitus during spring, and only less than 10 % of the dissected adults developed flight
muscle. Although adult S. venatus confluens have fully developed wings and make short flights
2-3 feet high in May and October in late afternoon (Kamm 1969), he believes walking is the
usual mode of spread in the field.
Hansen (1987) divided the instars of S. parvulus into three classes by larval head capsule
width: first instars < 0.6 mm, middle instars between 0.6 and 1.15 mm, and late instars < 1.15
mm. Four S. venatus confluens larvae reared on orchardgrass in the greenhouse had 5 instars
(Kamm 1969). Although there is no direct evidence shows the number of instars of S. venatus
vestitus from my larval head capsule data, at least 4 to 5 instars was speculated.
The second most abundant billbug species was the uneven billbug, S. inaequalis. It occurs
throughout the eastern United States and west to Texas, and north to central Florida (Peck and
28
Thomas 1998). The life cycle of S. inaequalis is barely known. Johnson-Cicalese et al. (1990)
found several S. inaequalis in the summer, but the population was more abundant in spring and
autumn in New Jersey, and was nearly equal in abundance with three other billbug species.
These result differ from our sampling data in Florida where there was a large adult S. inaequalis
population peak from mid-June to late August 2006 at West End Country Club. It is not clear
why the S. inaequalis population in 2007 was less than 2006, but there was still an obviously consistent population peak from June to August in 2007 which indicated one generation per year of S. inaequalis. The known hosts of S. inaequalis are bermudagrass, Kentucky bluegrass,
perennial ryegrass and fescues (Satterthwait 1931, Johnson-Cicalese and Funk 1990). Johnson-
Cicalese et al. (1990) suggested an equal pest status of S. inaequalis as the other three species in
New Jersey. However, a large number of S. inaequalis was only collected at West End Country
Club which suggest it may only cause damage in localized area of Florida.
Adult S. venatus vestitus can be found every month throughout the year in Florida with an extended oviposition period by females. The multiple overlapping generations makes control more difficult compared to the univoltine bluegrass billbug in northern states. However, among
the golf courses studied in Florida, serious billbug damage was only observed at one (LaGorce
Country Club in Miami Beach). This might indicate that most well managed bermudagrass turf
can tolerate certain levels of billbug attack. However, the irregularly distribution of
Sphenophorus spp. and overlapping life cycles at different locations in Florida need further studies. This study provides a fundamental knowledge of billbug species complex and seasonal activity in Florida for pest management.
29
Table 2-1. Total number of Sphenophorus spp. collected from four linear pitfall traps at each of four Florida golf courses during 24-hour sample periods from January 2006 to December 2007. Golf Course Species Gainesville West End LaGorce Card Sound Total S. apicalis 62 44 4 1 111 S. cariosus 5 25 0 0 30 S. cubensis 1 0 3 1 5 S. deficient 0 2 0 0 2 S. inaequalis 13 3,331 0 1 3,345 S. minimus 38 4 0 0 42 S. necydaloides 1 0 0 0 1 S. parvulus 0 2 0 0 2 S. pontederiae 1 1 0 0 2 S. venatus vestitus 2,073 428 8,898 3,641 15,040 Total 2,194 3,837 8,905 3,644 18,580
30
Table 2-2. Mean (±SEM) number of eggs per female S. venatus vestitus collected from three golf courses in Florida in 2007. Gainesville Country Club LaGorce Country Club Card Sound Country Club Month No. females No. eggs No. females No. eggs No. females No. eggs January 11 5.6 ± 0.8 15 3.3 ± 0.9 15 4.2 ± 0.8 February 22 3.7 ± 0.6 16 3.6 ± 0.6 13 3.0 ± 0.7 March 35 5.7 ± 0.4 18 5.1 ± 0.5 18 4.7 ± 0.6 April 89 5.5 ± 0.3 23 3.3 ± 0.5 20 2.4 ± 0.5 May 62 4.5 ± 0.4 20 1.8 ± 0.4 31 3.7 ± 0.3 June 62 4.4 ± 0.4 -- -- 20 3.2 ± 0.4 July 88 4.3 ± 0.3 20 1.8 ± 0.4 22 3.0 ± 0.5 August 81 4.8 ± 0.3 20 3.4 ± 0.6 20 3.2 ± 0.5 September 56 4.8 ± 0.3 20 2.6 ± 0.3 20 3.0 ± 0.4 October 63 6.0 ± 0.4 20 3.2 ± 0.4 20 3.4 ± 0.5 November 71 5.1 ± 0.4 42 4.2 ± 0.3 -- -- December 16 3.6 ± 0.5 20 4.1 ± 0.4 20 4.8 ± 0.4
31
Table 2-3. Mean (±SEM) number of eggs per female S. inaequalis collected at West End Country Club in 2007. Month No. females Mean¹ no. eggs January 10 2.0 ± 0.3 February 21 1.8 ± 0.2 March 31 2.3 ± 0.2 April 70 1.3 ± 0.1 May 48 1.3 ± 0.1 June 95 1.9 ± 0.1 July 92 2.3 ± 0.1 August 90 1.8 ± 0.1 September 60 2.4 ± 0.2 October 70 2.0 ± 0.1 November 46 1.4 ± 0.2 December 25 1.3 ± 0.2 ¹ Means within columns with different letters are statistically different at α = 0.05 (F = 6.52; df = 11, 669; P < 0.0001)
32
Table 2-4. Mean (± SEM) body lengths of male and female S. venatus vestitus at three golf courses in 2007. Male Body length Female Body length Mean # male body Mean # female body Golf Course max.- min. (mm) max.- min. (mm) length¹ (mm) length² (mm) Gainesville 6.8 - 8.3 7.8 - 9.5 7.7 ± 0.04a 8.5 ± 0.1a LaGorce 6.6 - 8.4 7.3 - 9.6 7.5 ± 0.10b 8.3 ± 0.1b Card Sound 6.8 - 8.4 7.1 - 9.3 7.5 ± 0.04b 8.1 ± 0.1b ¹ Means within columns with different letters are statistically different at α = 0.05 (F = 8.05; df = 2, 179; P = 0.0005) ² Means within columns with different letters are statistically different at α = 0.05 (F = 15.51; df = 2, 179; P < 0.0001)
33
70
60
50 collected 40
30 S. venatus vestitus 34
# 20
10
0 4-Jul 6-Jul 5-Jun 3-Oct 2-Jan. 17-Jul 31-Jul 18-Jul 6-Sep 5-Apr 7-Feb 5-Dec 2-Aug 8-Mar 7-Nov 16-Jan 30-Jan 8-May 20-Jun 20-Jun 2-May 24-Jan 10-Oct 24-Oct 17-Oct 31-Oct 16-Feb 27-Feb 11-Apr 25-Apr 12-Sep 25-Sep 19-Sep 18-Apr 21-Feb 19-Dec 14-Aug 30-Aug 15-Dec 22-Aug 14-Mar 27-Mar 21-Mar 21-Nov 14-Nov 28-Nov 23-May 16-May 31-May 2006 2007
Figure 2-1. Total number of S. venatus vestitus adults collected weekly from four linear pitfall traps at Gainesville Country Club.
700
600
d 500 collecte 400
300 35 S. venatus vestitus S. venatus # 200
100
0 9-Jan 7-Feb 4-Apr 12-Jul 26-Jul 11-Jul 8-Aug 6-Dec 7-Mar 7-Nov 3-May 13-Jun 29-Jun 13-Jun 28-Jan 17-Oct 23-Oct 21-Feb 24-Apr 19-Sep 13-Feb 10-Apr 17-Sep 22-Aug 20-Dec 10-amy 17-Aug 28-Aug 18-Dec 21-Mar 12-Mar 26-Mar 20-Nov 21-Nov 24-May 2006 2007
Figure 2-2. Total number of adult S. venatus vestitus obtained weekly from four linear pitfall traps at LaGorce Country Club.
180
160
140
120 collected
100
80
60 S. venatus vestitus S. venatus #
36 40
20
0 3-Jan 5-Jun 7-Feb 5-Apr 26-Jul 5-Apr 11-Jul 8-Aug 8-Mar 9-May 13-Jun 25-Jan 31-Jan 26-Jun 24-Oct 21-Feb 18-Apr 19-Sep 19-Feb 19-Apr 17-Sep 23-Aug 12-Dec 26-Dec 17-Aug 21-Mar 12-Mar 15-Nov 28-Nov 23-May 21-May 2006 2007
Figure 2-3. Total number of adult S. venatus vestitus trapped weekly at Card Sound Country Club.
160
140
d 120
100 collecte
80
60 # S. inaequalis 40
20 37
0 6-Jul 4-Jul 5-Jun 3-Oct 7-Feb 5-Apr 18-Jul 6-Sep 2-Jan. 17-Jul 31-Jul 2-Aug 5-Dec 8-Mar 7-Nov 24-Jan 2-May 20-Jun 20-Jun 16-Jan 30-Jan 8-May 17-Oct 31-Oct 10-Oct 24-Oct 21-Feb 18-Apr 19-Sep 16-Feb 27-Feb 11-Apr 25-Apr 12-Sep 25-Sep 22-Aug 15-Dec 14-Aug 30-Aug 19-Dec 21-Mar 14-Mar 27-Mar 14-Nov 28-Nov 21-Nov 16-May 31-May 23-May 2006 2007
Figure 2-4. Total number of S. inaequalis adults collected each week from four linear pitfall traps at West End Country Club.
4
3.5
3 observed
2.5 04/30/07' 11/11/07' 2 01/15/08' 1.5 02/07/08' S. venatus vestitus S. venatus
1
Mean # of # Mean of 0.5
0 4:00pm 8:00pm 12:00am 4:00am 8:00am 12:00pm 4:00pm
Figure 2-5. Mean number of S. venatus vestitus adults that were active on the soil surface of bermudagrass pots during a 24-hour period of observation.
38
35
d 30
collecte 25
# Eggs 20 # Larvae # Pupae 15 # Adults S. venatusvestitus 10
5 Number of of Number
0 5/24/2007 6/14/2007 7/5/2007 7/19/2007 07/26/2007
Figure 2-6. Number of life stage for S. venatus vestitus reared on pots of ‘Tifway’ bermudagrass in the greenhouse.
39
8
7
6 collected
5 # Eggs # Larvae 4 # Pupae 3 # Adults S. venatus vestitus S. venatus
2
Number of of Number 1
0 07/09/2007 07/23/2007 08/06/2007 08/20/2007 09/03/2007
Figure 2-7. Number of life stage for S. venatus vestitus reared on pots of ‘Empire’ zoysiagrass in the greenhouse.
40
18 16 14
e 12 10 8 6 Number of larva of Number 4 2 0 0.0 0.5 1.0 1.5 2.0 2.5 3.0 Head capsule width (mm)
Figure 2-8. Frequency distribution of head capsule width (mm) of S. venatus vestitus larvae collected from greenhouse rearing and eggs.
41
CHAPTER 3 EVALUATION OF BEMUDAGRASS AND ZOYSIAGRASS RESISTANCE TO Sphenophorus venatus vestitus
Billbug (Sphenophorus spp.) damage in turfgrass is often misdiagnosed as drought-stress or disease (Niemczyk and Shetlar 2000), leading to inappropriate management efforts. Early instar larvae are stem borers, late instar larvae are root feeders, and adults feed on stem tissue.
However, the late instar larvae are considered the most damaging stage (Satterthwait 1931). The most abundant and damaging billbug species in warm season turfgrass is the hunting billbug, S. venatus vestitus. Sphenophorus venatus vestitus has multiple generations with overlapping life stages each year in Florida (Chapter 2), which makes infestations of S. venatus vestitus difficult
to consistently manage with either preventive or curative insecticides. Its primary hosts are
bermudagrass (Cynodon dactylon (L.) Pers.) and zoysiagrass (Zoysia spp.).
An alternative, non-chemical control strategy is to develop cultivars that are more resistant
to billbug feeding damage. Resistance in different bermudagrass cultivars has been identified
against key arthropod pests (Quisenberry 1990) such as mites (Tashiro 1987), fall armyworm
(Spodoptera frugiperda J.E. Smith) (Quisenberry and Wilson 1985, Jamjanya and Quisenberry
1988, Croughan and Quisenberry 1989), and mole crickets (Scapteriscus spp.) (Reinert and
Busey 1984). Both non-preference and antibiosis to fall armyworm were identified in different
zoysiagrass cultivars (Chang et al. 1985). A host range studies on S. parvulus was done by
Kindler et al. (1983), they found only Kentucky blue grass, Poa pratensis L. showed significant infestation of S. parvulus larvae. Bermudagrass cultivars such as ‘Tifway’, ‘Tifdwarf’ and
‘Tifgreen’ have been identified as having high mite, Eriophyes cynodoniensis Sayed, resistance
(Tashiro 1987); Zoysia tenuifolia was also identified highly resistant to the Banks grass mite,
Oligonychus pratensis Banks (Busey 1982).
42
However, the first report of zoysiagrass cultivar resistance to S. venatus vestitus was by
Reinert (2001). The objective of this study was to document the impact to feeding damage by S.
venatus vestitus on a range of bermudagrass and zoysiagrass cultivars.
Materials and Methods
Damage potential of adult S. venatus vestitus on Fur Brmudagrass Cltivars
Plant material
Ten plugs (10.2 cm diameter) of each of the bermudagrass cultivars ‘Celebration’,
Tifdwarf, Tifeagle, and Tifway were obtained from established plots at the University of Florida
Plant Science Unit in Citra, FL, on 30 March 2007. Plugs were allowed to become established in pots (11.4 cm diameter) with native soil (sandy loam) in a greenhouse. Pots were fertilized with
113.5 g of Miracle-Gro® all purpose fertilizer (20-20-20) each week and irrigated daily.
Adult damage potential
Adult S. venatus vestitus were collected from linear pitfall traps at Gainesville Country
Club, FL, on 15 May 2007. Ten adult males or females of unknown age were placed onto the pot
of each bermudagrass cultivar (five pots per cultivar), and confined with a fine white mesh. After
2 weeks, pots were destructively sampled, adult survival per pot was determined, and the
location of male or female notching damage, number of notches per pot, notch length and width,
and diameter of damaged area were measured under a 10X dissecting microscope with a caliper.
The average daily temperature in the greenhouse was 16-30°C, average soil temperature was
19.4°C, light intensity was 9,688 lum/m2 with 14:10 hr (L:D). Data were analyzed using a two-
way ANOVA (SAS Institute 2000) to detect the effect of cultivar and billbug sex on adult S.
venatus vestitus damage potential. Means were compared using Tukey’s HSD test (P < 0.05).
43
Bermudagrass Cltivar Peference Test
Plant material
Cores of bermudagrass cultivars Celebration, Tifdwarf, Tifeagle, and Tifway were
obtained from the University of Florida Plant Science Unit in Citra, FL, on 24 August 2007.
Cores were planted into 8.9 cm diameter pots with native soil, fertilized weekly with 113.5 g of
Miracle-Gro® all purpose fertilizer (20-20-20) and irrigated daily. Grass height was maintained at about 5 cm. The test arena (22 × 22 × 10 cm) had fourteen holes (6 mm diameter) in the bottom to allow drainage, and an identical container with its bottom removed was glued upside down on top of it (Figure 3-1). One pot of each cultivar was randomly placed in the container, and the area between pots was filled with potting soil (Scotts Co. Gervais, OR) so the soil was 10 cm deep within and between pots.
Preference evaluation
Four of each unknown-aged adult male and female S. venatus vestitus were collected from
the U. F. Plant Science Unit in Citra, FL, on 20 November 2007 and released in the center of
each container between the pots within 24 hours. All adults were removed from containers 30
days after introduction (19 December 2007). All pots were evaluated on 9 - 10 January 2008 for
the total number of eggs and larvae in the stems or soil, and the total number of adult feeding
notches. Data were analyzed using a one-way ANOVA (SAS Institute 2000) to detect the effect
of variety on adult S. venatus vestitus preference. Means were compared using Tukey’s HSD test
(P < 0.05).
Zoysiagrass Cultivar Resistance to S. venatus vestitus
Plant material
Pots (7.6 cm diameter) of 17 zoysiagrass cultivars were established with sand in a
greenhouse in Gainesville, FL, for 90 days. Cultivars included ‘Belair’, ‘Cashmere’, ‘Cavalier’,
44
‘Compadre’, ‘Crowne’, ‘Diamond’, ‘El Toro’, ‘Emerald’, ‘Empire’, ‘Jamur’, ‘Palisades’,
‘Pristine Flora’, ‘Royal’, ‘Ultimate Flora’, ‘Zenith’, ‘Zeon’ and ‘Zorro’. Pots were fertilized with
113.5 g of Miracle-Gro® all purpose fertilizer (20-20-20) per week, irrigated as needed, and turf
was maintained at ~ 3.8 cm height.
Resistance evaluation
Adult S. venatus vestitus were collected from the pitfall traps at LaGorce Country Club in
Miami, FL, on 28 December 2006 and transported to the laboratory in Gainesville. Two males
and two females were placed onto each pot within 48 hours (five pots per cultivar), and confined
with a fine white mesh. Four weeks after adult caging, pots of each cultivar were rated for their
percentage of live cover (0-100%), color (1 = brown, 9 = dark green), density (1 = thin, 9 =
dense), overall quality (1 = poor, 9 = excellent), and the percentage of turf infested with the
armored scale Duplachionaspis divergens Green. Pots were also destructively sampled to recover
all life stages of S. venatus vestitus. Data were analyzed using a one-way ANOVA (SAS Institute
2000) to detect the effect of cultivar difference on adult S. venatus vestitus feeding potential and
oviposition. Means were separated using Tukey’s HSD test (P < 0.05).
This test was repeated with 18 cultivars (‘Meyer’ was added). Ten pots (7.6 cm diameter) per cultivar were planted in sand in November 2007, allowed to establish until January 2008, and maintained as previously described. Adult S. venatus vestitus were collected from the U.F. Plant
Science Unit in Citra, FL, on 11 January 2008, and two pairs of males and females were placed onto each pot within 24 hours. Each pot was encircled with clear plastic (15 cm high) to confine adults to the pot and allow normal light intensity and air flow. Four weeks after caging, pots of each cultivar were rated for their percentage of live cover, color, density, overall quality, and the percentage of turf infested with D. divergens. Pots were also destructively sampled to recover all
45
life stages of S. venatus vestitus. Data were analyzed using a one-way ANOVA (SAS Institute
2000) to detect the effect of cultivar difference on adult S. venatus vestitus feeding potential and
oviposition. Means were separated using Tukey’s HSD test (P < 0.05).
Results and Discussion
Damage Potential of Adult S. venatus vestitus on Four Bermudagrass Cultivars
Both male and female S. venatus vestitus caused damage, presumably by feeding, to the
bermudagrass stolons and rhizomes. The “notches” were 3.6 ± 0.3 mm below the nearest crown
for males and 4.0 ± 0.2 mm below for females in all varieties (Table 3-2). Notch length was
similar for both sexes (2.3 ± 0.2 mm for males, 2.4 ± 0.1 mm for females), and notch width was
identical (0.7 ± 0.03 mm). Females made more notches in pots of Tifway than in other cultivars
(F = 3.83; df = 3, 19; P = 0.0305), but males made a similar number of notches in pots of each cultivar (Table 3-1). Eggs were found in notches on stems or leaf sheath about 1.3 ± 0.1 mm wide. The diameter of the damaged area was significantly thicker in Tifway and Celebration than in Tifeagle and Tifdwarf (F = 8.2; df = 3, 39; P = 0.0003). It is possible that billbugs may preferentially attack cultivars with thicker stem diameters, such as Tifway and Celebration compared to Tifeagle and Tifdwarf (Kenworthy, 2008 personal communication). Thicker stems may indicate greater plant resources for their offspring. Turfgrass thatch thickness and density may also influence billbug populations by providing greater humidty and shade. For example, populations of S. parvulus were larger in areas with thicker thatch(Kindler and Spomer 1986).
Bermudagrass Cultivar Preference Test
One month after adult billbugs were caged onto pots, zero to two larvae and 0-25
notches occurred in pots of each cultivar. Tifway bermudagrass had the most of larvae and
notches recorded, and Tifeagle had the least (Table 3-2). The mean number of larvae was
significantly higher in Tifway than in Tifeagle (F = 3.49; df = 3, 39; P = 0.025). The mean
46
number of notches from adult feeding was significantly higher in Tifway than the other three
cultivars (F = 15.62; df = 3, 39; P < 0.0001).
Sphenophorus ventaus vestitus fed more, layed more eggs, and caused more damage on
Tifway bermudagrass than on the other bermudagrass cultivars tested. This may either indicate
greater preference for this cultivar or compensatory feeding. Further study to determine the host
quality and the impact on offspring survival, development time and fecundity are needed.
Currently there is no research documenting bermudagrass cultivars having resistance to S.
ventaus vestitus feeding. The genetic resistance among bermudagrass cultivars is unknown,
however, a broad range of grass species resistant to S. parvulus based on genetic differences was
identified (Asay et al. 1983). But they only rated the damage visually on each grass trial in the
field without recording number of notches on stem tissue or larvae present in the soil. Visual
damage may not have been the result of billbugs.
Zoysiagrass Cultivar Resistance to S. venatus vestitus
For the first run of this experiment ‘Diamond’ had the best performance four weeks after
adult billbug infestation for percent living coverage (F = 6.16; df = 16, 254; P < 0.0001), color
(F = 4.36; df = 16, 254; P < 0.0001), density (F = 17.48; df = 16, 84; P < 0.0001) and quality (F
= 7.63; df = 16, 254; P < 0.0001). ‘Belair’ performed the poorest for percent living coverage (F
= 6.16; df = 16, 254; P < 0.0001) and color (F = 4.36; df = 16, 254; P < 0.0001). ‘Compadre’
and ‘Empire’ had the least dense (F = 17.48; df = 16, 84; P < 0.0001). Belair and Compadre had
the poorest turf quality (F = 7.63; df = 16, 254; P < 0.0001) (Table 3-3). Compadre, Diamond,
‘Emerald’, and ‘Royal’ were more susceptible to the scale, D. divergens (F = 14.98; df = 16, 254;
P < 0.0001). Notches made by adults were 0.8 to 3.9 mm long and 0.4 to1.0 mm wide across cultivars (Table 3-4).
47
For all cultivars, zero to eight eggs + larvae and one to thirteen notches were found in pots
4 weeks after adults were caged (Table 3-4). The mean number of notches, and the mean number of eggs + larvae were not significantly different among two zoysiagrass species or the hybrids.
Regardless of species, the mean number of eggs and larvae combined was significantly higher in
‘Crowne’, ‘El Toro’, and ‘Jamur’ than other cultivars (F = 1.91; df = 16, 84; P = 0.0338), but the mean number of notches was not significantly different among cultivars.
After evaluating the second test, 0-12 eggs and larvar, 4-38 notches were found per pot
(Table 3-5). Among Z. japonica cultivars, the mean number of notches, and the mean number of eggs + larvae combined were not significantly different. Among Z. matrella cultivars, the mean number of notches (F = 4.52; df = 5, 29; P = 0.0048), and eggs plus larvae combined were significantly higher in ‘Cashmere’ than other cultivars (F = 3.5; df = 5, 29; P = 0.0162). Among hybrid cultivars, the mean number of notches, and the mean number of eggs + larvae combined were not significantly different. Regardless of cultivar, the mean number of notches, and the mean number of eggs and larvae combined was significantly higher in Z. japonica than other species (F = 24.02; df = 2, 89; P < 0.0001) (F = 14.5; df = 2, 89; P < 0.0001), respectively.
Regardless species, significantly more notches occurred in ‘Meyer’, ’Zenith’, Compadre, and
‘Palisades’ than in other cultivars (F = 5.01; df = 17, 89; P < 0.0001). The mean number of eggs and larvae combined was significantly higher in Meyer, Crowne, Compadre, ‘Zenith’, and El
Toro than other cultivars (F = 2.81; df = 17, 89; P = 0.0012).
Reinert (2001) reported that four cultivars of Z. matrella (Diamond, ‘DALZ 9601’,
‘Cavalier’ and ‘Royal’) are resistant to S. venatus vestitus, and ‘Meyer’ is the most susceptible among nine cultivars. In this study, cultivars with the highest number of eggs + larvae such as
Compadre, Crowne, ‘Jamur’, Meyer, and Zenith were all in Z. japonica. Z. matrella cultivars
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generally produced fewer S. venatus vestitus eggs or larvae. with less offspring. Total number of damage was also higher in Z. japonica cultivars than in Z. matrella cultivars. However, billbugs feeding more on cultivars of Z. japonica did not mean they were more susceptible since I did not evaluate the nutrient level of each cultivar. Thus this feeding behavior could be compensatory.
These findings plus the rating data indicate that cultivars of Z. matrella have better resistance against S. venatus vestitus feeding and oviposition.
The reason I found mostly larvae in the first evaluation and mostly eggs in the second time evaluation remains unknown. I speculate that the warmer temperature of the greenhouse in the first time evaluation speed up egg hatch to larvae. In addition, genetic resistance in grass cultivars to insect pest may be influenced by soil fertility and cultural conditions in which the grass is grown (Quisenberry 1990). Therefore, to eliminate all the potential factors that might affect the results, such as grass resources, pot size, amount of fertilizer, I did both bermudagrass and zoysiagrass trials in our greenhouse rather than in the field. However, two different populations of S. ventaus vestitus were used from different locations (Gainesville and Miami) in the test, and were used in different seasons, which may have influenced test results.
To conclude, cultivars of both bermudagrass and zoysiagrass appear to have varying levels of susceptibility to S. venatus vestitus. Turfgrass managers may be able to reduce their pesticide use by selecting less susceptible cultivars when installing, renovating, or repairing turfgrass areas.
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Table 3-1. Damage between male and female S. venatus vestitus on four bermudagrass cultivars in the greenhouse. Diameter of Billbug Mean no. notches Notch length Notch width Cultivar damaged area gender (±SEM)1/pot (mm) (mm) (mm) Celebration Male 21.2 ± 2.1 2.0 ± 0.2 0.7 ± 0.04 1.5 ± 0.1 Tifdwarf Male 29.6 ± 3.7 1.8 ± 0.1 0.8 ± 0.03 0.9 ± 0.04 Tifeagle Male 24.8 ± 3.2 1.8 ± 0.1 0.6 ± 0.02 0.7 ± 0.02 Tifway Male 22.0 ± 4.8 2.3 ± 0.2 0.7 ± 0.04 1.2 ± 0.04
Celebration Female 23.2 ± 2.1b 2.4 ± 0.2 0.8 ± 0.04 1.3 ± 0.1 Tifdwarf Female 26.8 ± 1.8b 1.7 ± 0.1 0.8 ± 0.03 1.1 ± 0.03 Tifeagle Female 26.2 ± 2.8b 2.0 ± 0.1 0.6 ± 0.03 0.7 ± 0.02 Tifway Female 34.8 ± 3.1a 2.4 ± 0.1 0.7 ± 0.03 1.3 ± 0.04 1 Means within columns with different letters are statistically different at α = 0.05 (F = 3.83; df = 3, 19; P = 0.0305)
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Table 3-2. Mean (±SEM) number of notches and surviving larvae per pot on four bermudagrass cultivars in the greenhouse preference test. Cultivar Mean1 no. larvae/pot Mean2 no. notches /pot Celebration 0.4 ± 0.2b 6.3 ± 0.9b Tifdwarf 0.3 ± 0.2b 6.1 ± 2.0b Tifeagle 0b 2 ± 0.4b Tifway 0.7 ± 0.2a 15.7 ± 1.9a 1 Means within columns with different letters are statistically different at α = 0.05 (F = 3.49; df = 3, 39; P = 0.025) 2 Means within columns with different letters are statistically different at α = 0.05 (F = 15.62; df = 3, 39; P < 0.0001)
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Table 3-3. Ratings (±SEM) of seventeen zoysiagrass cultivars after 4 weeks of adult S. venatus vestitus infestation. Species/Cultivar Percent live cover 1 Color (1-9)2 Density (1-9)3 Quality (1-9)4 % D. divergens5 Zoysia japonica Belair 34 ± 4.3e 4.5 ± 0.4d 4.2 ± 0.4cde 2.9 ± 0.2e 10.3 ± 2.9c Compadre 50 ± 3.1bcde 6.1 ± 0.4abcd 3.3 ± 0.3e 2.9 ± 0.2e 45.3 ± 8.3a Crowne 60.7 ± 3.3abcd 6.1 ± 0.3abcd 5.5 ± 0.3bcd 4.6 ± 0.4bcde 2.0 ± 0.8c El Toro 41.7 ± 7.8de 5.8 ± 0.1abcd 6.7 ± 0.3ab 3.8 ± 0.6cde 4.3 ± 1.0c Empire 60.0 ± 4.7abcd 6.4 ± 0.3abc 3.3 ± 0.3e 3.2 ± 0.3de 18.3 ± 3.8bc Jamur 66.0 ± 3.1abc 6.3 ± 0.4abc 5.5 ± 0.4bcd 5.0 ± 0.4abcd 9.7 ± 2.6c Palisades 68.7 ± 4.6ab 6.5 ± 0.3ab 4.2 ± 0.3cde 4.6 ± 0.3bcde 6.3 ± 1.4c Ultimate Flora 57.3 ± 3.5abcd 6.5 ± 0.4ab 5.5 ± 0.4bcd 4.2 ± 0.4bcde 7.3 ± 1.3c Zenith 43.3 ± 4.9cde 4.9 ± 0.4bcd 4.1 ± 0.4de 3.6 ± 0.5de 15.0 ± 5.4c Zoysia matralla Cashmere 58.7 ± 3.6abcd 5.4 ± 0.3abcd 5.9 ± 0.3bc 4.9 ± 0.2abcd 2.3 ± 0.7c Cavalier 47.7 ± 6.9bcde 4.8 ± 0.7cd 6.9 ± 0.3ab 3.7 ± 0.6cde 19.7 ± 6.3bc Diamond 79.6 ± 3.6a 6.9 ± 0.3a 7.7 ± 0.4a 6.7 ± 0.3a 40.7 ± 6.5ab Pristine Flora 63.3 ± 3.6abcd 5.9 ± 0.3abcd 6.2 ± 0.4ab 5.1 ± 0.3abcd 5.3 ± 1.4c Royal 70.0 ± 4.6ab 6.8 ± 0.2a 7.1 ± 0.3ab 5.7 ± 0.3ab 54.3 ± 8.2a Zeon 56.7 ± 6.0bcde 6.1 ± 0.3abcd 7.6 ± 0.3a 5.5 ± 0.4abc 11.7 ± 3.8c Zorro 58.3 ± 4.2abcd 6.3 ± 0.2abc 7.0 ± 0.3ab 5.0 ± 0.4abcd 10.3 ± 3.0c Hybrid Emerald 50.0 ± 3.5bcde 6.6 ± 0.3a 5.5 ± 0.4bcd 3.9 ± 0.3bcde 50.7 ± 6.9a 1 Means within columns with different letters are statistically different at α = 0.05 (F = 6.16; df = 16, 254; P < 0.0001). 2 Means within columns with different letters are statistically different at α = 0.05 (F = 4.36; df = 16, 254; P < 0.0001). 3 Means within columns with different letters are statistically different at α = 0.05 (F = 17.48; df = 16, 84; P < 0.0001). 4 Means within columns with different letters are statistically different at α = 0.05 (F = 7.63; df = 16, 254; P < 0.0001). 5 Means within columns with different letters are statistically different at α = 0.05 (F = 14.98; df = 16, 254; P < 0.0001)
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Table 3-4. Mean number (±SEM) of S. venatus vestitus eggs and larvae, and characteristics of adult notches on 17 zoysiagrass cultivars, after 1 month of adult confinement. # Mean no. eggs Mean no. Notch Length Notch Width Species/Cultivar and larvae/pot notches /pot (mm) (mm) Zoysia japonica Belair 0 1.4 ± 0.5 3.1 ± 0.7 0.6 ± 0.1 Compatibility 0 1.0 ± 0.4 1.5 ± 0.4 0.6 ± 0.1 Crowne 1.6 ± 0.7 2.6 ± 1.1 1.7 ± 0.4 0.6 ± 0.1 ElToro 1.2 ± 0.6 1.6 ± 0.7 1.3 ± 0.2 1.0 ± 0.1 Empire 0.8 ± 0.5 2.0 ± 0.9 1.2 ± 0.3 0.6 ± 0.1 Jamur 1.4 ± 0.9 1.6 ± 1.2 3.6 ± 0.7 1.0 ± 0.2 Palisades 0 0.6 ± 0.2 1.1 ± 0.2 0.5 ± 0.03 Utimate Flora 0.4 ± 0.4 1.2 ± 0.7 1.3 ± 0.2 0.7 ± 0.1 Zenith 0 0.2 ± 0.2 0.8 0.4 Zoysia matrella Cashmere 0.8 ± 0.6 2.4 ± 1.2 3.3 ± 0.9 0.5 ± 0.1 Cavalier 0.4 ± 0.2 1.2 ± 0.8 1.4 ± 0.2 0.5 ± 0.04 Diamond 0 0.6 ± 0.4 0.8 ± 0.1 0.4 ± 0.03 Pristine Flora 0 1.4 ± 0.7 3.6 ± 0.9 0.6 ± 0.1 Royal 0.2 ± 0.2 2.2 ± 1.1 2.6 ± 1.1 0.6 ± 0.1 Zeon 0 1.2 ± 0.4 1.6 ± 0.4 0.5 ± 0.02 Zorro 0.2 ± 0.2 1.0 ± 0.3 3.9 ± 1.2 0.6 ± 0.1 Hybrid Emerald 0 0.4 ± 0.4 1.4 ± 0.8 0.4 ± 0.1
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Table 3-5. Mean number (±SEM) of S. venatus vestitus eggs and larvae, and adult notches on 18 zoysiagrass cultivars, after one month of adult confinement. Species/Cultivar Mean no. eggs and larvae/pot Mean1 notches/pot Zoysia japonica Belair 0.8 ± 0.4 5.6 ± 1.9abcd Compadre 2.4 ± 0.9 6.2 ± 1.5abc Crowne 2.6 ± 0.7 3.2 ± 0.9abcd ElToro 1.8 ± 0.6 3.4 ± 0.7abcd Empire 0.8 ± 0.4 5.6 ± 1.9abcd Jamur 1.2 ± 0.5 4.8 ± 0.9abcd Meyer 3.0 ± 0.9 6.8 ± 1.2ab Palisades 1.0 ± 0.5 5.8 ± 0.7abcd Utimate Flora 0.2 ± 0.2 0.8 ± 0.4 Zenith 2.6 ± 1.7 7.6 ± 1.3a Zoysia matrella Cashmere 1.0 ± 0.3 5.4 ± 1.1abcd Cavalier 0.4 ± 0.2 1.4 ± 0.2cd Diamond 0 1.6 ± 0.8bcd Pristine Flora 0 1.8 ± 0.4bcd Royal 0.2 ± 0.2 1.6 ± 0.5bcd Zeon 0.2 ± 0.2 1.0 ± 0.4cd Zorro 0 2.6 ± 1.0abcd Hybrid Emerald 0 1.0 ± 0.3cd 1 Means within columns with different letters are statistically different at α = 0.05 (F = 5.01; df = 17, 89; P < 0.0001)
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Figure 3-1. Sphenophorus venatus vestitus preference test for bermudagrass cultivars.
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CHAPTER 4 EFFECT OF ENDOPHYTE LEVEL IN PERENNIAL RYEGRASS ON THE SURVIVAL AND DEVELOPMENT OF Sphenophorus venatus vestitus
Endophytic fungi and phytophagous insects acquire nutrients from sharing the same host plant and interacting with one another (Wilson and Carroll 1997). Studies have documented that the endophytic fungus Neotyphodium lolii Latch associated with grasses such as perennial ryegrass (Lolium perenne L.) or tall fescue (Festuca arundinacea Schreb) can have enhanced resistance to herbivory through the production of alkaloids (Prestidge and Gallagher 1988,
Kunkel et al. 2003). Some insects can detect endophyte infected grass and avoid feeding on it, while those that do feed on it suffer reduced fitness, survival and fecundity (Johnson-Cicalese
1997).
Endophytes occur in 13 genera of grasses including bluegrass, bentgrass, fescue, and ryegrass, and affect 40 insect species from six different orders (Johnson-Cicalese 1997).
Endophytic tall fescue was more effective against first and second instar white grubs than third instars (Potter et al. 1992, Koppenhöfer et al. 2003). Ahmad et al. (1986) reported that a reduction of bluegrass billbug (Sphenophrous parvulus Gyllenhal) larval population density and feeding damage was associated with endophytic perennial ryegrass. Subsequently, Richmond et al. (2000) reported that, in general, visual damage and the larval numbers of S. parvulus decreased as the proportion of endophytic perennial ryegrass increased. Another field trial in
New Jersey indicated that endophytic tall fescue provided a high level of resistance to the damage of four billbug species (Murphy et al. 1993). Endophyte-enhanced turfgrass resistance also affects populations of sod webworm (Murphy et al. 1993), Argentine stem weevil,
Listronotus bonariensis (Kuschel) (Prestidge 1988), and gall-forming insects, Besbicus mirabilis
Kinsey (Wilson and Carroll 1997). However, this is the first report on the effect of pure and overseeded endophytic perennial ryegrass on the hunting billbug (S. venatus vestitus Chittenden).
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Materials and Methods
Sphenophorus venatus vestitus Survival, Development, and Damage on Four Endoyphytic Perennial Ryegrass Cultivars
To assess the direct effect of endophytic perennial ryegrass on the survival and
development of S. venatus vestitus, a greenhouse experiment was conducted. The cultivars
‘Citation Fore’ (76% endophyte), ‘Catalina II’ (72%), ‘Brightstar SLT’ (80%), and ‘Caparral II’
(6%) (Scotts Company) were seeded at a rate of 29.4 kg / 1,000 m2 with 80% sand mixed with
20% top soil in Plastic pots (11.7 cm diameter) on 17 November 2007. The endophyte level of
each cultivar was evaluated by the Agrinostics Company. Each treatment was replicated five
times in a randomized complete block design. Miracle-Gro® all purpose fertilizer (113.5 g, 20-
20-20) was applied to each pot weekly and pots were irrigated daily. Each pot was infested with
two male and two female adult S. venatus vestitus collected from ‘Tifway’ bermudagrass at the
U.F. Plant Science Research Unit in Citra, FL, on 10 January 2008. Pots were encircled up to 15
cm above the turf height with a clear plastic tube to prevent adult escape. Turfgrass damage was
visually rated (1 = little damage, 9 = severe damage), and adult survival, and the number of eggs
and larvae found in stem tissue or soil were determined after 2 weeks (24 January 2008). Data were analyzed using a one-way ANOVA (SAS Institute 2000) to detect the effect of different
cultivars on adult S. venatus vestitus damage potential and oviposition. Means were compared
using Tukey’s HSD test (P < 0.05).
Impact of Overseeding Two Bermudagrass Cultivars with Endophytic Perennial Ryegrass on S. venatus vestitus Survival, Development, and Damage
To assess the effect of overseeding two bermudagrass cultivars with a perennial ryegrass
cultivar with a high percentage of endophyte on the survival and development of S. venatus
vestitus, a greenhouse experiment was conducted. Cores (10.2 cm diameter) of Tifway and
‘Tifeagle’ bermudagrass were obtained from the U.F. Plant Science Research Unit in Citra, FL,
57
and planted into 11.7 cm diameter plastic pots on 27 April 2007, and were allowed to establish
with native soil (sandy loam). Five pots of each bermudagrass cultivar were overseeded on 17
November 2007 at a rate of 50.5 kg / 1,000 m2 with ‘Citation Fore’, a perennial ryegrass cultivar
with 76% endophyte. Five additional pots of each bermudagrass cultivar not overseeded.
Miracle-Gro® all purpose fertilizer (113.5 g, 20-20-20) was applied to each pot weekly and pots were irrigated daily. Each pot was infested with two adult male and two female S. venatus vestitus collected from the U.F. Plant Science Research Unit in Citra, FL, on 10 January 2008.
Each pot was encircled up to 15 cm above the turf height with a clear plastic tube to prevent adult escape. Pots were destructively evaluated and the total number of eggs and larvae found in stem tissues and soil, and number of adult notches made, were recorded after 2 weeks (28
January 2008). Data were analyzed using a two-way ANOVA (SAS Institute 2000) to detect the effect of different cultivars and overseeding on adult S. venatus vestitus damage potential and oviposition. Means were compared using Tukey’s HSD test (P < 0.05).
Overseeded Bermudagrass Field Trial
Due to limitations in seed availability, two different perennial ryegrass cultivars were used in the field study. A cultivar with a low endophyte level (SR4500, 33% endophyte) and a cultivar with a high endophyte level (SR4420, 78% endophyte) were overseeded onto plots (1.83 × 1.83
m) of ‘Tifway’ bermudagrass at the U.F. Plant Science Research Unit in Citra, FL, on 9
November 2006 at a rate of 50.4 kg/1,000 m2 using a Scott’s Proturf professional drop spreader.
Control plots were not overseeded. Bermudagrass height was ~1 cm and thatch was ~1.4 mm
thick. Each ryegrass treatment was replicated ten times, but there was only space for five control
plots (25 total plots), in an unbalanced randomized complete block design. About 950 S. venatus vestitus and 450 S. inaequalis adults were collected from Gainesville golf courses and released onto the plots to establish a population during late summer and fall of 2006, before overseeding
58
occurred. Turf and soil cores (10.2 cm diameter, 8 cm depth) were removed from each plot on 19
November 2006 (3 cores per plot), 20 January (2 cores per plot) and 2 April 2007 (5 cores per plot) and destructively examined for all billbug life stages. Data were analyzed with two-way
ANOVAs (SAS Institute 2000), and if significant, means were separated by Tukey’s HSD test
(SAS Institute 2000).
Results and Discussion
Sphenophorus venatus vestitus Survival, Development, and Damage on Four Endoyphytic Perennial Ryegrass Cultivars
No eggs, larvae, or adult notching damage were found in the pots of any of the four
ryegrass cultivars after 2 weeks of being infested with adult S. venatus vestitus, regardless of
endophyte level. The adult survival of each cultivar was 95% in ‘Citation Fore’, 80% in
‘Catalina II’, 85% in ‘Brightstar SLT’, and 95% in ‘Caparral II’. Females were not dissected to
examine the number of eggs in their ovaries after 2 weeks of caging. Only some grass blades
turned yellowish and shrank, especially in ‘Caparral II.’ Perennial ryegrass is considered a host
for S. venatus vestitus (Jonhson-Cicalese and Funk 1990), although I did not measure the stem
diameter, I assumed perhaps the stem systems were not developed or thick enough to allow first
instar feeding, so oviposition by female S. venatus vestitus had not occurred. Given the lack of
notching damage, adults may have been repelled or not induced to feed and oviposit. Ants and
spiders can detect alkaloids from their potential prey’s integument (Montllor et al. 1991,
Schaffner et al. 1994), so perhaps S. venatus vestitus can also sense the presence of alkaloids
from endophyte-infected turfgrass cultivars.
Impact of Overseeding Two Bermudagrass Cultivars with Endophytic Perennial Ryegrass on S. venatus vestitus Survival, Development, and Damage
This is the first study to examine the effect of overseeding with endophyte-enhanced
perennial ryegrass on S. venatus vestitus, for potential use as a management tool in the southern
59
United States. However, Murphy et al. (1993) demonstrated that endophyte-enhanced tall fescue
(Festuca arundinacea Schreb) reduces the feeding damage of S. venatus vestitus and S. minimus.
From all ryegrass cultivars in this study, zero to three larvae, and three to 40 notches were found in pots after 2 weeks of being infested with adults. Eggs were not found in all pots. The mean number of notches and larvae were significantly higher in non-overseeded Tifway pots than in other cultivars (F = 32.44; df = 3, 19; P < 0.0001) and (F = 10.43; df = 3, 19; P = 0.0005), respectively. Tifeagle overseeded with endophytic perennial ryegrass had the least damage and no larvae were found (Table 4-1). Regardless of cultivar, the mean number of notches and larvae were significantly higher in control pots than in overseeded pots (F = 25.41; df = 1, 19; P <
0.0001) and (F = 18.78; df = 1, 19; P = 0.0004), respectively. It is unclear whether adults avoided feeding and ovipositing on overseeded bermudagrass, or if eggs and early instar larvae died in the overseeded pots. Similarly, in the northern U.S., overseeding Kentucky bluegrass
(Poa pratensis L.) with endophytic perennial ryegrass can reduce S. parvulus larval populations and damage (Richmond et al. 2000).
Overseeded Bermudagrass Field Trial
Despite releasing over 1,000 Sphenophorus spp. adults into the bermudagrass plots before
overseeding was done, only one first instar was found in a stolon from a control plot in the
November 2006 sample, one mature larva was found in each of two control plots and one in an
SR 4500 (low endophyte) plot in the January 2007 sample, and one mature larva was found in a
control plot and one from SR 4500 in the April 2007 sample. Grass roots and stems were healthy
in all turf cores; no obvious damage or notches were visible. The number of billbugs collected
from these samples was too low to detect treatment differences. It is possible that a large enough
billbug population did not develop on the plots, and that the number of cores taken at each
sampling date was insufficient. However, results from caging only two pairs of S. venatus
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vestitus in greenhouse pot trials demonstrates that adults can cause significant turfgrass damage and lay several eggs in small areas. The field plots were surrounded by other potential non- overseeded turfgrass areas, so it is also possible that adults moved out of the desired area (they were not confined with cages). Other overseeding field studies were done in areas with a history of billbug infestation, rather than newly infested areas (Murphy et al. 1993, and Richmond et al.
2000). Turfgrass height could also affect female billbug oviposition choice and/or larval survival, if it impacts stem or root diameter and density.
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Table 4-1. The effect of overseeding with endophytic perennial ryegrass on two bermudagrass cultivars on S. venatus vestitus damage potential and oviposition Mean no. notches/pot Mean no. larvae/pot Cultivar Treatment (±SEM)1 (±SEM)2 Tifway Control 31.2 ± 3.3a 1.8 ± 0.4a Tifeagle Control 18.4 ± 1.2b 0.8 ± 0.4b Tifway Overseeded 13.2 ± 1.0b 0b Tifeagle Overseeded 5.6 ± 0.9c 0b 1 Means within columns with different letters are statistically different at α = 0.05 (F = 32.44; df = 3, 19; P < 0.0001). 2 Means within columns with different letters are statistically different at α = 0.05 (F = 10.43; df = 3, 19; P = 0.0005).
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APPENDIX A EVALUATION OF BEMUDAGRASS RESISTANCE TO Sphenophorus inaequalis
Materials and Methods
Damage Potential of Adult S. inaequalis on Four Bermudagrass Cultivars
Plant material
Ten plugs (10.2 cm diameter) of each of the bermudagrass cultivars ‘Celebration’,
‘Tifdwarf’, ‘Tifeagle’, and ‘Tifway’ were obtained from established plots at the University of
Florida Plant Science Unit in Citra, FL, on 30 May 2006. Plugs were allowed to become established in pots (11.4 cm diameter) with native soil (sandy loam) in a greenhouse. Pots were fertilized with 113.5 g of Miracle-Gro® all purpose fertilizer (20-20-20) each week and irrigated daily.
Adult damage potential
Adult S. inaequalis were collected from linear pitfall traps at West End Country Club in
Gainesville, FL, on 8 August 2006. Ten unaged adult males or females were placed onto the pot
of each bermudagrass cultivar (five pots per cultivar), and confined with a fine white mesh. After
2 weeks, pots were destructively sampled, adult survival per pot was determined, and the
location of male or female notching damage, number of notches per pot, notch length and width
were recorded. In the S. inaequalis test the average daily temperature in the greenhouse was 21-
32°C, average soil temperature was 21°C, light intensity was 10,764 lum/m2 with 14:10 hr (L:D).
Data were analyzed using a two-way ANOVA (SAS Institute 2000) to detect the effect of variety
and billbug sex on adult damage potential. Means were compared using Tukey’s HSD test (P <
0.05).
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Results and Discussion
Damage Potential of Adult S. inaequalis on Four Bermudagrass Cultivars
The notches caused by S. inaequalis were 3.0 ± 0.3 mm below crown for males and 4.1 ±
0.3 mm below for females in all varieties (Table 3-1). Average notch length was 2.0 ± 0.2 mm for males and 1.9 ± 0.1 mm for females. Average notch width was 0.7 ± 0.04 mm for males and
0.7 ± 0.03 mm for females. The mean number of notches from both male and female feeding was significantly higher in Tifdwarf than other cultivars (males: F = 35.1; df = 3, 19; P < 0.05; females: F = 16.1; df = 3, 19; P < 0.05). The mean number of notches caused by females feeding across cultivars was significantly higher than males (F = 5.81; df = 1, 39; P < 0.05) (Table 3-1).
In the bermudagrass variety trial, S. inaequalis tended to feed more on Tifdwarf bermudagrass than other cultivars, and female S. inaequalis also fed more than males under a no- choice environment. The reason why Tifdwarf was the most susceptible variety to S. inaequalis feeding among the four bermudagrass varieties is not clear understood. One reason might be the less resistance from the cultivar itself. Tashiro (1987) reported a high mite resistance in the bermudagrass cultivars Tifdwarf and Tifway. However, Tifdwarf apparently has less resistance against to S. inaequalis feeding comparing to other cultivars. It is reasonable that females cause more notches than males because the needs of energy for ovary development and oviposition.
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Table A-1. Damage between male and female S. inaequalis on four bermudagrass cultivars in the greenhouse Mean1, 2 no. Diameter of Billbug notches Notch length Notch width damaged area % adult Cultivar gender (±SEM)/pot (mm) (mm) (mm) survival Celebration Male 16.2 ± 1.5b 1.7 ± 0.1 0.6 ± 0.02 1.0 ± 0.04 70 Tifdwarf Male 34.6 ± 2.0a 1.6 ± 0.1 0.6 ± 0.02 0.7 ± 0.02 86 Tifeagle Male 18.6 ± 1.7b 1.0 ± 0.1 0.4 ± 0.01 0.7 ± 0.02 84 Tifway Male 12.2 ± 1.4b 2.0 ± 0.2 0.7 ± 0.04 1.2 ± 0.06 64 Celebration Female 20.8 ± 1.5b 1.7 ± 0.1 0.7 ± 0.03 1.1 ± 0.04 70 Tifdwarf Female 42.4 ± 3.2a 2.5 ± 0.2 0.8 ± 0.03 1.0 ± 0.03 82.5 Tifeagle Female 20.4 ± 2.1b 1.0 ± 0.1 0.4 ± 0.01 0.7 ± 0.02 76 Tifway Female 28.8 ± 3.1b 1.9 ± 0.1 0.7 ± 0.03 1.2 ± 0.03 82 1 Means within columns with different letters are statistically different at α = 0.05 (F = 35.1; df = 3, 19; P < 0.05). 2 Means within columns with different letters are statistically different at α = 0.05 (F = 16.1; df = 3, 19; P < 0.05).
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BIOGRAPHICAL SKETCH
Ta-I Huang was born, grew up and attended school in Taipei, Taiwan. He studied forestry in the Chinese Culture University, where he obtained his bachelor’s degree in 2003. He moved to the United States and studied at PALS English program at Rutgers University in 2005. He attended to the University of Florida, Entomology and Nematology Department in spring 2006 as a Graduate Research Associate in the landscape entomology lab under Dr. Buss’s supervision where he started studying toward his master’s degree.
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