Beauveria bassiana and Elisa Determination of Neonicotinoids to Improve Management of Listronotus maculicollis

The “annual bluegrass weevil” (Listronotus maculicollis) became resistant to synthetic pyrethroid insecticides (bifenthrin and cyhalothrin) in several adult weevil populations from Connecticut, Rhode Island, and Massachusetts in 2007-09, and management of this insect has become increasingly complex. Annual bluegrass weevil continues to be a serious pest of Poa annua L. (Poaceae) and bentgrasses (Agrostis spp) on many golf courses in mid-Atlantic and northeastern United States and eastern Canada. Adults chew notches on grass blades and at the juncture of leaves and stems. However, adult feeding has little effect on plant vitality. Early instars feed inside plant stems and late instars on plant crowns. The first generation larvae, which usually become apparent in late May or early June, typically cause the most severe damage. During July and August damage caused by second generation larvae is usually less extensive, especially if good control of the overwintering generation was obtained. In most cases, adequate control of the insect has been achieved through the use of pyrethroid applications targeting adult weevils as they emerge from overwintering sites and before they begin to lay eggs. However, if a population is resistant to pyrethroids, alternative controls are required to prevent damage. My research objectives were to evaluate the entomopathogenic fungus Beauveria bassiana for control of L. maculicollis and how neonicotinoid insecticides can best be used to manage this increasingly serious pest. While pyrethroids remain the preferred choice of many golf course superintendents for managing this species, resistance has forced some superintendents to incorporate other strategies. Some of the new strategies include: (1) the use of a pyrethroid or chlorpyrifos early against overwintering adults; (2) neonicotinoid/pyrethroid combinations (Aloft, Allectus) during peak adult emergence to control adults and first generation larvae; (3) primarily preventative larvicidal compounds (chlorantraniliprole (Acelepryn), neonicotinoids) for early instars; and (4) curative larvicidal compounds (trichlorfon (Dylox), spinosad (Conserve), indoxacarb (Provaunt), chlorpyrifos, pyrethroids) for control of fourth and fifth instars. Some locations may need to use one or more of these strategies to prevent turf damage and resistance development. It is imperative that the timing of treatments coincide with various life stages (adults, early or late instar larvae) to maximize chemical efficacy. This is particularly important for the systemic compounds (neonicotinoids / chlorantraniliprole) to insure there is sufficient chemical in the xylem for maximum effectiveness. If treatment strategies 1-3 are not effective, a curative larvicidal compound may need to be applied to prevent damage. Finally, since all subsequent generations come from the overwintering adults, it is imperative that a superintendent control those adults and any larvae that they produce (1 generation).

). This insect species was first seen damaging turfgrass in Connecticut in 1931 and by the late 1950s and early 1960s was responsible for severe damage on golf courses in the state (Britton 1932, Tashiro 1976).
Adult L. maculicollis chew notches on grass blades at the juncture of leaves and stems.
Adult damage is not as severe as larval feeding. Larval feeding can result in extensive turf damage and death since they feed at the plant crown. Where larval densities exceed 450 per 929 cm 2 (1 ft 2 ), injury to golf course greens, collars, and fairways is common Fourth generation pyrethroids provided excellent control of weevils in the 1990s and early 2000s. These products were principally used to target adult weevils as they colonized turf after overwintering in areas surrounding tees, greens, and fairways. In 2005, the first indications of diminished pyrethroid effectiveness were reported (Vittum 2005). In 2009 the first study to confirm pyrethroid resistance was published (Ramoutar 2009a), and two subsequent studies (Ramoutar et al. 2009b; further confirmed pyrethroid resistance. Alternative controls are needed to manage this serious pest. Beauveria bassiana (Balsamo) Vuillemin is an important biological control agent for many insect pests (Fargues and Luz 2000;Furlong and Groden 2001;Quintela and McCoy 1998). In this study we evaluated a commercially available formulation of B. bassiana against L. maculicollis adults in Petri dish assays and simulated field studies with turfgrass plugs both with and without neonicotinoid insecticides and a nutrient source for B. bassiana called MycoMax. We concentrated on adult control because instars 1-3 are protected inside plant stems and can only be controlled with systemic insecticides and by the time 4 th and 5 th instars emerge, the majority of damage has occurred. Furthermore, adult population densities are more readily monitored than larval stages.

METHODS
Petri Dish Assays. Adult weevils were collected from different golf courses and placed on 9 cm diameter filter paper discs in 100 × 15 mm Petri dishes treated with various dosages of Beauveria bassiana strain GHA and its "inert" carrier oil (BotaniGard, Laverlam International, Butte, MT), the "inert" carrier oil alone, and neonicotinoid insecticides and combinations with BotaniGard and its "inert" carrier oil applied in 0.5 or 1 ml water. Controls consisted of treating filter paper discs with 1 ml of water and adding adult weevils. Petri dishes were wrapped in parafilm to maintain humidity and prevent weevils from escaping. Assays were rated for adult mortality for up to 10 d. In the first set of assays, we evaluated the highest label dosage of B.
In a second assay, we evaluated three neonicotinoid insecticides: imidacloprid (Bayer Environmental Science, Research Triangle Park, NC), clothianidin (Valent U. S. A. Corp, Walnut Creek, CA) and dinotefuran (PBI -Gordon, Kansas City, MO) at label dosages with and without one-tenth the label dosage of B. bassiana strain GHA and its "inert" carrier oil and 88.7% of one-tenth the label dosage of the "inert" carrier oil alone for adult mortality 1, 3, 5, and 7 d after treatment. One experiment was conducted with five replicates with weevils from Westerly, RI (10 weevils per replicate, 5 replicates, 50 weevils per treatment).
Turf Plug Assays. In another series of experiments, we evaluated the highest label dosage of BotaniGard (25.46 liters/ha); however, mortality was not high enough to be able to recommend it as a control option. Therefore, we compared the highest label dosage and 4× the highest label dosage (101.84 liters/ha) to explore a dosage response, and to determine whether mortality would be comparable to standard synthetic insecticides. Controls consisted of treating 5.72 cm diameter turfgrass plugs with water only and adding ten weevils. Turfgrass plugs were treated using a CO 2 powered sprayer equipped with an 8002EVS TeeJet nozzle (Spraying Systems Co., Wheaton, IL). After treatment, turfgrass plugs were placed in 147 ml plastic cups where the weevils were added and then contained with a 1 mm mesh screen. Three assays using weevils from two locations (Westerly, RI and Norwich, CT) (10 weevils per plug, 4 replicates, 120 weevils per treatment) were rated for adult mortality 7, 10 and 14 d after treatment and combined for analysis.
In another set of turf plug assays, adult weevils (5 per plug, 4 replicates, 20 weevils per treatment) were collected from Westerly and Pawtucket, RI and placed on turfgrass plugs treated with the highest label dosage (25.46 liters/ha) of B. bassiana strain GHA in its "inert" carrier oil both with and without 5 and 20% MycoMax (a sweet whey designed to be a nutrient source for B. bassiana obtained from Dr. Scott Costa, Univ. of VT) in volumes of water equal to 815 liters/ha (2 gal/1,000 ft 2 ).
Controls consisted of treating turfgrass plugs with water only and adding five weevils.
Statistical Analysis. Percent mortalities were transformed by taking the arcsine of the square root of the proportion before ANOVA and mean separation via Tukey's HSD test (SAS version 9.2). Untransformed means and errors are shown in figures.
There was significant mortality of adults in Petri dish assays with label dosages of imidacloprid with one-tenth the label dosage of BotaniGard (2.54 liters/ha) 1 d after treatment in Petri dish assays (F = 40.18; df =6,24; P < 0.01) (Fig. 3). Clothianidin, imidacloprid and dinotefuran alone at the label dosage also caused significant mortality of adults 3 d after treatment (Figs. 2, 3 and 4).

DISCUSSION
Petri dish assays at the highest label dosage (25.46 liters/ha) and the "inert" carrier oil filtered 2× at that dosage were very effective in causing mortality of adult weevils at 24 h. This indicates that mortality within 24 h was the result of the oil and not infection from B. bassiana strain GHA. When using oil formulations of entomopathogenic fungi there needs to be assurance that insecticidal activity is not the result of the oil carrier (Goettel and Inglis 1997). Typical infection with entomopathogenic fungi such as B. bassiana will normally take several days to exert lethal effects which we did notice beginning on day five. Cowles et al. (2000) used leaf dip bioassays with twospotted spider mites, Tetranychus urticae Koch and demonstrated the toxicity of trisiloxane surfactants, also considered inert ingredients. Toxicity was influenced by the leaf dip method which exaggerated the degree of wetting and indicated that high toxicity from surfactants was likely only in extremely wetted applications with high humidity. Although we were using "inert" carrier oil as a treatment and not a surfactant there was high mortality associated with a higher degree of wetness. This suggests that the Petri dish assay treatments of BotaniGard and its carrier oil using 1 ml of water may be effectively drowning the weevils similar to the activity of surfactants against T. urticae reported by Cowles et al. (2000).
The influence of moisture and humidity was also evident in the difference between treatments applied in volumes of 0.5 versus 1.0 ml of water. The "inert" carrier oil was ineffective when applied with 0.5 ml of water where the Petri dish was not as wet. However, when BotaniGard was applied in Petri dishes in 0.5 ml of water, significant mortality was evident starting at 5 d and increased at 7 and 10 d after treatment. This suggests that B. bassiana did begin to infect and kill adult L. maculicollis and that humidity and moisture were still high enough for infection even with 0.5 ml of water. This is supported by other experimental work that showed moisture was critical in effectiveness of B. bassiana strain GHA. Fargues and Luz (2000) found that the pathogenic activity of B. bassiana to Rhodnius prolixus Ståhl was highly dependent on the moisture conditions and to a lesser extent on the temperature conditions. Their results showed a critical threshold of relative humidity between 95.5 and 97%.
One of the most encouraging results was the effect of one-tenth the label dosage of BotaniGard or the "inert" carrier oil with neonicotinoids (imidacloprid, clothianidin and dinotefuran). We noticed in other assays that neonicotinoids have a quick knockdown effect on adults; however, adults normally recover within 24 hours.
Immobilizing adults for 24 hours may allow B. bassiana to overcome the insects' defense mechanisms or the "inert" carrier oil is drowning adults. There was a significant difference in mortality between one-tenth the label dosage of BotaniGard and its "inert" carrier oil beginning on day five after treatment. Furlong and Groden (2001) found that significant synergism occurred in all instances where Colorado potato beetle, Leptinotarsa decemlineata (Say), larvae were exposed to imidacloprid before or simultaneously with B. bassiana treatment. They suggested that the synergism probably involves changes in the insect's physiology that affects successful cuticular penetration or the initial proliferation of B. bassiana hyphal bodies within the host hemocoel. Quintela and McCoy (1998) found that the addition of imidacloprid to soil significantly impaired movement of larval Diaprepes abbreviates (L.). When either B.      (Vittum et al. 1999). This insect species was first seen damaging turfgrass in Connecticut in 1931 and by the late 1950s and early 1960s it was responsible for severe damage on golf courses in the state (Britton 1932, Tashiro 1976).
Adult L. maculicollis chew notches on grass blades at the juncture of leaves and stems.
Adult damage is not as severe as larval feeding, which can result in extensive turf damage and death since they feed at the plant crown. Where larval densities exceed 450 per 929 cm 2 (1 foot 2 ), injury to golf course greens, collars, and fairways is common  (Vittum 2005). In 2009 the first study to confirm pyrethroid resistance was published (Ramoutar et al. 2009a). Two subsequent studies (Ramoutar et al. 2009b; 2010) further confirmed pyrethroid resistance.
We tested the hypothesis that treating P. annua plants early in the season would have enough neonicotinoid insecticide (either clothianidin or imidacloprid) in their tissue to control overwintering adult L. maculicollis as they feed. A second goal was to determine the concentrations of clothianidin and imidacloprid in P. annua tissue necessary to kill larvae and how long these toxic levels remained in plant tissue. Two turf plugs from each plot were placed in modified Berlese funnels similar to the method used by Diaz (2008) (Fig. 1). Five milliliters of glycerin was used in the bottom collection containers to hold larvae. Funnel containers were checked up to 14 d after collection and larvae were removed and placed in vials of 70% ethyl alcohol until head capsule width could be measured. Head capsule width was measured using a binocular microscope fitted with an eyepiece reticle at 63× magnification to determine larval instars. The third plug from each plot was used for collection of grass clippings used for ELISA determination of neonicotinoid concentration. At least 0.5 g (fresh weight) of grass clippings was cut from the turf plug then placed in a labeled plastic bag and stored at -20˚C until analysis.
Imidacloprid lasted longer at higher levels in plant tissue at Baltic, CT, however, there were no significant differences in treated versus control plots of 1-3, 4-5, or 1-5 instar larvae (Figs. 12,13,14). The same patterns were seen in plots treated in Westerly, RI .

DISCUSSION
Adults are not killed by the concentrations of neonicotinoids we found in P. annua tissue. This does not support the hypothesis that superintendents should "arm" P. annua plants very early in the season with neonicotinoid insecticides to control adult weevils which are emerging from overwintering sites as they begin to feed. We expected to see more adult mortality from the combination products that contained bifenthrin. However, bifenthrin has a Koc value of 236,610 ml/g (Pesticide Properties Database, 2013), which means that bifenthrin is tightly adsorbed to organic matter and, once dried on P. annua tissue, it may not be available for control unless it is rewetted via irrigation and/or dew. Bifenthrin's estimated Henry's constant of 7.2 X 10 -3 atmm 3 /mole indicates that volatilization from moist soil surfaces may occur (Bifenthrin Technical Fact Sheet). Rewetting of P. annua tissue did not occur in our turf plug assay.
The long soil half-lives of clothianidin and imidacloprid, 545 and 191 d respectively (Pesticide Properties Database, 2013), also led to the hypothesis that if these materials were applied early in the season to control overwintering adults, the concentrations inside P. annua tissue would still be high enough later in the season to control first generation larvae. This does not appear to be the case. First through third instars feed inside P. annua stems while 4 th and 5 th instars feed on plant crowns. Koppenhofer et al. (2012) found that applications of clothianidin or imidacloprid between April 15 and May 3 rd provided an average of 54 and 48% control respectively, whereas applications between May 18 th and June 10 provided averages of 64 and 78% control respectively. It appears that we applied these products earlier than optimal timing to demonstrate any significant control. This information is important for managing this pest.
There were 4 th and 5 th instars present as early as 24 and 26 April (Figs. 10,13,16,19) which may have been controlled by the bifenthrin in the Aloft and Allectus treatments. This is supported by the fact that larval densities were higher in the Arena treated plots despite the fact that the clothianidin levels were higher to begin with (treated with 448 g ai/hectare) versus larval densities in Aloft treated plots, which was treated with 280 g ai/hectare. The levels of clothianidin were also consistently higher in P. annua tissue in Arena versus Aloft treated plots. The same goes for the Merit and Allectus treated plots which were treated with (448 and 280 g ai/hectare respectively).
The levels of imidacloprid were also consistently higher in P. annua tissue in Merit versus Allectus treated plots.
In 10 of 12 analyses larval counts were lower where bifenthrin was one of the treatment components . Although some companies are guaranteeing season-long control, our data did not show any season-long control at either Baltic, CT (Fig. 21) or Westerly, RI (Fig. 22). Koppenhofer et al. (2012) analyzed data from 1,064 field experiments with various insecticides for annual bluegrass weevil control. Of these, 57 were Merit applications (various formulations) with imidacloprid rates between 140 and 560 g ai/ha. The majority of applications were with either 337 g ai/ha (32 applications) or 448 g ai/ha (14 applications). Four of the 337 g ai/ha applications showed zero percent control, even though they were applied when I would expect some level of control of larvae. Similarly, at total of 49 Arena applications (46 were 50 WDG and 3 were 0.5G formulations) with clothianidin rates between 168 and 449 g ai/ha were analyzed. The majority of these applications were either 224 g ai/ha (13 applications) or 449 g ai/ha (15 applications). Two of the 280 g ai/ha, one 337 g ai/ha, and one 449 g ai/ha applications showed zero percent control, again, when we would expect some level of control. Koppenhofer et al. (2012) found that several populations of L. maculicollis could be labeled as resistant to pyrethroids, organophosphates, neonicotinoids, indoxacarb, and bifenthrin / neonicotinoid combination products. If the populations of L. maculicollis at Baltic, CT and Westerly, RI were among those that demonstrated multiple resistance, this could explain the lack of control in our experiments. Fig. 5. Percent mortality (mean + SE) of adult weevils from Westerly, RI after feeding for 7 or 14 days on plugs of P. annua containing 94, 100, 783, or 1,136 ng clothianidin / g tissue. Means followed by the same letter are not significantly different (P = 0.05, Tukey's HSD test). DAT = days after treatment. DOF = days of feeding.