VOL. 34, NO. 2 SOUTHWESTERN ENTOMOLOGIST JUN. 2009

Efficacy of Entomopathogenic Fungi in Suppressing Weevil, caryae (Coleoptera: ), in Commercial Pecan Orchards

David I. Shapiro-Ilan1, Ted E. Cottrell1, Wayne A. Gardner2, Robert W. Behle3, Bill Ree4, and Marvin K. Harris4

Abstract. The pecan weevil, Curculio caryae (Horn), is a key pest of , Carya illinoinensis (Wangenh.) K. Koch. The entomopathogenic fungi Beauveria bassiana (Balsamo) Vuillemin and Metarhizium anisopliae (Metschnikoff) Sorokin are pathogenic to and are being developed as microbial control agents for pecan weevil. One approach to suppressing pest populations and the resultant damage might be to apply entomopathogenic fungi when adult pecan weevils are emerging from the soil. Here we report the efficacy of B. bassiana (GHA strain) and M. anisopliae (F52 strain) applied to trees in orchards at three locations: Byron, GA, Fort Valley, GA, and Comanche, TX. At Fort Valley, treatments included B. bassiana as an oil-based spray with a UV-protective screen applied to the trunk, M. anisopliae applied as an impregnated fiber band stapled onto the trunk, and a nontreated check. At Byron, GA, we compared the B. bassiana trunk treatment to a nontreated check. Treatments at the Texas location were the B. bassiana trunk application, M. anisopliae applied as a trunk band and as a soil drench, and a nontreated check. At each location, weevils were trapped and transported to the laboratory for 15 to 17 days post-treatment to record mortality and mycosis. At both Georgia locations, B. bassiana caused •80% mortality and mycosis, which was significantly greater than mortality observed in the check (”33%); mortality and mycosis in the M. anisopliae treatment at Fort Valley did not differ from that observed in the check. In Texas, due to insufficient replication in plots, statistical comparison among treatments was not possible. However, mean percentages of mortality of pecan weevils after 7 and 14 days were 38 and 55% in the check, 75 and 88% in the B. bassiana-treated plots, and 57 and 75% in the M. anisopliae- treated plots. These results indicate potential for B. bassiana trunk sprays to suppress adult pecan weevil. Future research is needed to determine if the approach contributes to economic levels of crop protection.

______1USDA-ARS, SE Fruit and Tree Research Laboratory, Byron, GA 31008 2Department of Entomology, University of Georgia, Griffin Campus, 1109 Experiment Street, Griffin, GA 30223 3USDA-ARS-NCAUR, Peoria, IL 61604 4Department of Entomology, Texas A&M University, College Station, TX 77843

This article reports the results of research only. Mention of a proprietary product does not constitute an endorsement or recommendation for its use by USDA.

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Introduction

The pecan weevil, Curculio caryae (Horn), is a key pest of pecans, Carya illinoinensis (Wangenh.) K. Koch, throughout the southeastern US as well as portions of Texas and Oklahoma (Payne and Dutcher 1985). The have a 2- or 3-year life cycle (Harris 1985). Adult weevils emerge from soil in late July-August to feed on and oviposit in developing nuts. Once larval development is completed within the nut, 4th instars drop to the soil and burrow to a depth of 8-25 cm, and form a soil-cell to overwinter. The following fall approximately 90% of the larvae pupate and spend the next 9 months in the soil as adults before emerging. The remaining 10% of the population spend 2 years in the soil as larvae and emerge as adults in the 3rd year. Current recommendations for controlling pecan weevil consist mainly of above-ground applications of chemical insecticides (e.g., carbaryl) targeting adults in the canopy (Harris 1999, Hudson et al. 2006). Application of chemical insecticides is recommended every 7-10 days during peak emergence of pecan weevils (Ree et al. 2005, Hudson et al. 2006). Because of problems associated with resurgence of aphids and mites that often result from chemical applications (Dutcher and Payne 1985), as well as other environmental and regulatory concerns, developing alternative control strategies is desirable. Microbial pesticides such as the entomopathogenic fungi Beauveria bassiana (Balsamo) Vuillemin and Metarhizium anisopliae (Metschnikoff) Sorokin are potential alternatives (Shapiro- Ilan 2003, Shapiro-Ilan et al. 2008). Both the larval and adult stages of pecan weevil are susceptible to infection by these agents (Tedders et al. 1973; Gottwald and Tedders 1983; Harrison et al. 1993; Shapiro-Ilan et al. 2003, 2008, 2009). Prior research indicated that many emerging pecan weevils either crawl or fly to the trunk (Raney and Eikenbary 1968, Cottrell and Wood 2008). By exploiting this behavior, significant mortality may be obtained by applying the fungus to soil surrounding the trunk, or directly to the trunk, thereby targeting the insects before they enter the canopy to feed and oviposit. Indeed, recently, Shapiro-Ilan et al. (2008) reported that suppression of pecan weevil was affected by the method of applying B. bassiana; spraying the fungus directly on the trunk resulted in greater mortality of pecan weevil relative to direct application to soil. Additionally, another approach to applying fungus to the trunk, i.e., wrapping non-woven fiber bands impregnated with M. anisopliae around the trunk, caused significant mortality of emerging pecan weevils (Shapiro-Ilan et al. 2009). The objective of this study was to investigate the potential of applications of these fungi to suppress pecan weevil in commercial pecan orchards. We applied entomopathogenic fungi in pecan orchards at three weevil-infested locations: Byron, GA, Fort Valley, GA, and Comanche, TX.

Materials and Methods

‘Stewart’, ‘Frotcher’, and ‘Van Demon’ varieties of pecan trees >150 years old in loamy-sand soil were used at Byron, GA. The Fort Valley, GA, orchard consisted of loamy-sand soil with ‘Stuart’ and ‘Schley’ trees approximately 60 years old. The Texas location was approximately 16 km north of Comanche, and consisted of native variety trees in a fine sandy-loam soil; trees varied in age and size (all non-pecan tree species had been removed 30 years earlier.)

112 Beauveria bassiana (GHA strain), i.e., Botanigard®, used in all field experiments was obtained from Emerald BioAgriculture Corporation (Butte, MT) as an emulsifiable oil formulation containing 2 x 1013 conidia per 946-ml container. This strain (GHA) has been labeled for use in controlling pecan weevil. Metarhizium anisopliae (F52 strain), formulated into fiber bands, was produced at the USDA- ARS Research Station in Stoneville, MS, based on procedures described by Hajek et al. (2006), and refrigerated while shipped to Byron, GA. M. anisopliae conidia for application to soil were produced through a biphasic system (liquid blastospore suspension poured into sterile rice medium in bags) based on the method of Leland et al. (2005). All fungal material was stored at approximately 4oC and used within 2 months of receipt. Before application, viability of conidia was verified on agar as described by Goettel and Inglis (1997), and >80% viability was deemed acceptable. At the two orchards in Georgia, the experiments were in randomized complete block designs with six blocks (tree rows) containing each treatment and a nontreated check. Each plot consisted of a single tree. At the Texas location, the experiment also had six trees per treatment and check, but treatments were not randomized; each treatment (or check) consisted of a single large-block in a demonstration-style plot (note that because of lack of randomization among plots the data from the orchard in Texas were not analyzed statistically). The fungi were applied according to Shapiro-Ilan et al. (2008, 2009). B. bassiana was applied as a trunk treatment at all locations, whereas (because of a shortage of material) M. anisopliae trunk-bands were applied at Fort Valley, GA, and Comanche, TX, but not at Byron, GA. For trunk application with B. bassiana, 236.5 ml of BotaniGard ES (5 x 1012 conidia) and 100 ml of SoyScreen oil were mixed with sunflower oil (ConAgra Foods, Irving, CA) to reach a total volume of 1 liter. A CO2-charged backpack sprayer (310.3 kPa, Spray Systems Co., Wheaton, IL) with a cone nozzle (5500-X8 adjustable conejet) was used to apply the mixture to approximately 1.5 m of the bottom part of the trunk. The SoyScreen was added as a UV-protecting adjuvant (Compton and Laszlo 2002). The M. anisopliae fungus bands (approximately 45 x 3 cm) were attached horizontally around the circumference of the trunk by stapling the ends and middle of each; six or seven bands were required to encircle the trunk. Two rings of bands were attached to each tree, one approximately 123 cm above the ground and the other 135 cm above the ground. At Comanche, TX, an aqueous ground application of M. anisopliae also was applied around each tree at a rate of 5 x 1012 conidia per plot (hence, the rate-per-unit area was approximately 6.4 x 1010 conidia per m2); M. anisopliae was mixed with approximately 30.3 liters of water and 0.01% Silwet L-77, (Loveland Industries, Inc., Greeley, CO) and applied via watering can to a radius of 5 m around each trunk. Applications were on 15 August 2006 in Georgia and 24 August in Texas. Efficacy of the treatments was estimated for naturally emerging pecan weevils. Adult pecan weevils were collected in Circle traps attached to pecan trunks (Mulder et al. 2003). This is a passive trap that captures insects crawling up the trunk. The traps were made of 1.5-mm wire mesh with an open area approximately 61 cm wide facing toward the soil (to collect ascending weevils) and tapering up to a removable top. Traps were placed on the trunk so the bottom of the trap was approximately 100 cm above the soil surface. The top of the trap (the removable one-way cone portion where insects accumulate) was placed on the trap approximately 24 hours before each sample date. Four or five traps were placed on each tree so the entire circumference of the trunk was covered. At the locations in

113 Georgia, pecan weevils were collected 1, 3, 8,10, and 15 days after treatment, and in Texas, weevils were collected daily starting 5 days after treatment through 17 days post-treatment (except no weevils were collected 9, 10, or 11 days post- treatment). On each day pecan weevils were trapped in the field, the insects captured in each trap were placed in separate plastic bags and transported to the laboratory to determine amounts of fungal infection. All pecan weevils were placed individually into 30-ml plastic cups (3-4 cm i.d., 3.5 cm deep) with a 3-cm cotton wick moistened with approximately 2.1 ml of tap water. Cups were placed in plastic boxes (28 x 15 x 9.5 cm deep) organized by block (for the Georgia locations), and incubated in darkness at 25oC. For the Georgia locations, after 14 days of incubation, the percentage of pecan weevil mycosis per plot was estimated by examining the cadavers for signs of fungal infection (Goettel and Inglis 1997, Shapiro-Ilan et al. 2003), and the percentage of total pecan weevil mortality (mycosis plus other causes) was recorded. For the Texas location, pecan weevil mortality and mycosis were recorded after 7 and 14 days of incubation. For the Georgia locations, treatment effects averaged during the 15-day sampling period were determined with repeated measures analysis using conservative degrees of freedom (Cochran and Cox 1957, Steel and Torrie 1980, SAS 2002). If the F-value was significant, treatment differences were further elucidated with Student-Newman-Keuls’ test. All percentage data were transformed by arcsine of the square root before analysis (non-transformed means are presented). The alpha level for all statistical tests was 0.05. Because of lack of sufficient replication (as indicated previously) data from Comanche, TX, were not analyzed statistically. Average (± SD) mortalities and mycoses were recorded for each treatment.

Results

At Byron, GA, total percentage of pecan weevil mortality (mycosis and other causes) was greater with the B. bassiana trunk treatment than the nontreated check (F = 11.80; df = 1, 3; P = 0.041) (Fig. 1A). Similarly, mycosis in the B. bassiana treatment was greater than the check (F = 18.32; df = 1, 3; P = 0.023) (Fig. 1B). At Fort Valley, GA, total percentage of pecan weevil mortality was greater with the B. bassiana trunk treatment than the M. anisopliae trunk-band treatment or the nontreated check (which were not different from each other) (F = 5.88; df = 2, 8; P = 0.027) (Fig. 2A). Mycosis in the B. bassiana treatment was also greater than the M. anisopliae treatment or the nontreated check (F = 21.16; df = 2, 8; P = 0.0006) (Fig. 2B). In Georgia, pecan weevil mortality in the B. bassiana treatments was 89 and 100% at Byron and Fort Valley, respectively, whereas mortality did not surpass 33% in the check plots (Figs. 1 and 2). At Byron, GA, 90 weevils were captured during the experimental period (36 in the control plots and 54 in the B. bassiana plots), and at Fort Valley, GA, 111 pecan weevils were captured (36 in the check, 16 in B. bassiana plots, and 59 in the M. anisopliae plots). At Comanche, TX, the average (± SD) percentage total mortalities after 7 days of incubation were 38.4 ± 37.2, 75.0 ± 38.8, and 57.3 ± 13.4 in the check, B. bassiana, and M. anisopliae plots, respectively. Percentages of weevils exhibiting signs of mycosis after 7 days of incubation were 8.6 ± 16.1, 22.9 ± 36.6, and 16.5 ± 11.0 in the check, B. bassiana, and M. anisopliae plots, respectively. After 14 days of incubation, average (± SD) percentage total mortalities were 54.9 ± 34.7, 84.5±

114 19.4, and 74.8 ± 8.7 in the check, B. bassiana, and M. anisopliae plots, respectively. Percentages of weevils exhibiting mycosis after 14 d of incubation were 12.1 ± 12.9, 42.7 ± 33.2, and 29.7 ± 11.4 in the check, B. bassiana, and M. anisopliae plots, respectively. The total numbers of weevils captured during the experiment were 61 in the check, 16 in the B. bassiana, and 657 in the M. anisopliae plots.

A

B

Fig. 1. Mean (± SE) percentage adult Curculio caryae mortality (A), and weevils exhibiting signs of mycosis (B) following application of Beauveria bassiana (Bb) in a commercial pecan orchard at Byron, GA. Bars represent mean percentages (± SE) averaged over a 15-day sampling period in 2006. Approximately, 5 x 1012 conidia were applied per tree. Fungal treatments were sprayed on the trunk. Check (Control) = not treated. Different letters above bars indicate statistically significant differences (SNK test, α = 0.05).

115 A

B

Fig. 2. Mean (± SE) percentage adult Curculio caryae mortality (A) and mycosis (B) following applications of Beauveria bassiana and Metarhizium anisopliae in a commercial pecan orchard at Fort Valley, GA. Bars represent mean percentages (± SE) averaged over a 15-day sampling period in 2006. Beauveria bassiana (Bb) was sprayed on the trunk at a rate of 5 x 1012 conidia per tree. Metarhizium anisopliae (Ma) was applied as fungal impregnated fiber bands attached to the tree trunk. Check (Control) = not treated. Different letters above bars indicate statistically significant differences (SNK test, α = 0.05).

Discussion

Direct application of B. bassiana to the tree trunk, and M. anisopliae applied as a trunk-band (with or without additional ground application of conidia) have been

116 shown to kill emerging pecan weevil adults (Shapiro-Ilan et al. 2008, 2009), yet this is the first report that included both approaches, and in one replicated study (Fort Valley, GA) compared them directly. Additionally, this is the first report of such fungal treatments applied to a natural population of pecan weevil in a commercial orchard. In replicated studies at Byron and Fort Valley, GA, application of B. bassiana to the trunk killed many pecan weevils. Our results with B. bassiana are consistent with those of Shapiro-Ilan et al. (2008), thereby confirming the potential for applying B. bassiana to the trunk to suppress pecan weevil. Indeed, similar to our observations, in a 2-year study, Shapiro-Ilan et al. (2008) observed >75% weevil mortality during a 15-day period. Furthermore, greater levels of mortality when B. bassiana was applied to the trunk were observed compared with ground applications (particularly if the application was followed by cultivation). In contrast to B. bassiana, pecan weevil mortality and mycosis in replicated plots treated with M. anisopliae bands were not different from results in check plots. These results are also in contrast to observations in other systems, e.g., M. anisopliae or Beauveria brongniartii (Sacc.) Petch, fiber bands controlled various cerambycid (Shimazu et al. 1995, Dubois et al. 2004, Hajek et al. 2006). Additionally, our results with M. anisopliae seem to be in contrast with an earlier study by Shapiro-Ilan et al. (2009) in which M. anisopliae applied as a trunk band caused significant infection in pecan weevil. However, results in the prior study (Shapiro-Ilan et al. 2009) were also variable, e.g., total pecan weevil mortality was different from the check in the first year of the study but not in the second (although mycosis was different both years). Thus, the lack of significant differences observed in the current study may be an extension of the natural variability of the approach, which may depend on varying environmental conditions, potency of particular fungal batches, etc. Given that the innate virulence of M. anisopliae (F52) to pecan weevil in laboratory studies was similar to that of B. bassiana (GHA) (Shapiro-Ilan et al. 2009), the poor or variable results observed with field applications of M. anisopliae thus far have been unexpected. We cannot determine the cause of the differences in field efficacy between the two fungal species because a variety of factors have differed between the two treatments. Possibly the differences were caused by varying production or formulation methods, or varying sensitivity to environmental conditions. Alternatively, the method of application (trunk spray versus trunk band) may be responsible for differences in efficacy. Conceivably, the trunk spray is a superior approach because the area of the trunk treated (and thus the duration of exposure) is greater than the trunk-band approach. Additional studies such as comparing the two fungal species using the same method of application are needed to determine the cause of differential efficacy. Results from the demonstration in Texas are generally consistent with the replicated experiments in Georgia. Although we could not statistically analyze the data from Texas, or directly compare the experiments in Georgia and Texas, the trends seem to be similar in that pecan weevil mortality in B. bassiana plots was greater than in M. anisopliae plots. However, the few weevils captured in the Texas B. bassiana plot may lend less weight to the comparison (because of the potential for variability when weevils are few); clearly, substantially more pecan weevils were captured in the M. anisopliae plots than elsewhere. One might argue that the few weevils in some of the replicated plots in Georgia are also problematic, e.g., the captures at the Fort Valley, GA B. bassiana plots. However, the fact that results at Fort Valley, GA (i.e., with B. bassiana) were consistent with those at Byron, GA, as

117 well as with previous studies (Shapiro-Ilan et al. 2008), and the fact that the data were separated statistically, support our experimental conclusions. Possibly, the great numbers of weevils killed following application of B. bassiana to trunks could be developed further as part of a management program for pecan weevils. The number of weevils killed in this study, however, may not necessarily translate into economic levels of crop protection. Given that it can take •7 days for B. bassiana to kill pecan weevils (Shapiro-Ilan et al. 2004), the weevils are likely to cause at least some feeding or oviposition damage to the nuts before the fungus takes effect (Criswell et al. 1975). Furthermore, our evaluation is only an estimation of potential (rather than actual) efficacy in the field. This is because our analysis was based on pecan weevil mortality following transport to controlled environmental conditions; thus, we do not know how many of the weevils might have survived if they had remained in the field. Therefore, additional research is needed to determine if application of B. bassiana to trunks contributes to economic control of the target pest. Additionally, research is warranted to determine if other approaches to fungus application, instead of or in addition to application to trunks, would be advantageous. For example, applications targeting larvae or other soil- dwelling stages may contribute significantly to suppression of pecan weevil. Furthermore, integration or combinations with other control agents such as entomopathogenic nematodes (which are pathogenic to pecan weevil [Shapiro-Ilan et al. 2006]) or chemical insecticides (which may also enhance activity of B. bassiana [Quintela and McCoy 1998]) should be investigated.

Acknowledgment

We thank Ann Amis, Rebekah Auman, Danny Davis, Wanda Evans, Kathy Halat, Roger Laster, Grace Lathrop, Paul Loller, and Hal Peeler for technical assistance; Jarrod Leland for providing M. anisopliae; and Frank Funderburk, Frank Hiley, and Robbie Robertson for use of their orchards. This research was supported in part by a USDA-SARE grant (Project #LS03-153). References Cited

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