BIOLOGICAL CONTROLÐMICROBIALS Natural Occurrence of Entomopathogens in Pacific Northwest Nursery Soils and Their Virulence to the Black Vine , sulcatus (F.) (Coleoptera: )

DENNY J. BRUCK1

USDAÐARS, Horticultural Crops Research Laboratory, 3420 NW Orchard Avenue, Corvallis, OR 97330

Environ. Entomol. 33(5): 1335Ð1343 (2004) ABSTRACT The black vine weevil Otiorhynchus sulcatus (F.) is the primary pest of Þeld and container-grown woody ornamentals in the PaciÞc Northwest (PNW). These studies were conducted to determine the natural occurrence of soil-borne entomopathogens in PNW nursery soils and determine their virulence to black vine weevil. Soil samples were collected JulyÐSeptember of 2002 from Þeld-grown woody ornamental nursery stock in Oregon, Washington, and Idaho. Sample collection in each state took place in the major nursery production areas. A total of 280 samples was collected (Oregon, 170; Washington, 50; Idaho, 60). Entomopathogens were isolated using insect baiting (nematodes and fungi) as well as semiselective media (fungi). Bacillus thuringiensis Berliner was isolated through sodium acetate selection. Soil-borne entomopathogenic fungi occur widely throughout the major nursery production areas in the PNW. The entomopathogenic fungi Metarhi- zium anisopliae (Metchnikoff) Sorokin, (Balsamo) Vuillemin, and Paecilomyces tenuipes (Peck) Samson were isolated. An entomopathogenic nematode (Steinernema oregonense Liu and Berry) and B. thuringiensis were also isolated. Of the 30 fungal isolates bioassayed, all but one was pathogenic to last-instar black vine weevil. None of the B. thuringiensis isolates collected were pathogenic to adult black vine weevil. The S. oregonense that were collected only infected a single black vine weevil larvae at 15 and 22ЊC. Pathogens collected from this soil survey will serve as a source of potential biological control agents for black vine weevil.

KEY WORDS Beauveria bassiana, anisopliae, Bacillus thuringiensis, Steinernema ore- gonense, soil survey

SOIL-INHABITING ENTOMOPATHOGENIC FUNGI (Harrison Adults cause mainly esthetic damage to ornamentals and Gardner 1991, Bing and Lewis 1993, Chandler et by feeding on leaves. Eggs are deposited on the soil al. 1997, Bidochka et al. 1998, Klingen et al. 2002, surface, and larvae cause severe damage by feeding on Shapiro-Ilan et al. 2003), nematodes (Homminick the roots of plants (Smith 1932). of the genus 2002) and bacteria (Martin and Travers 1989) have Otiorhynchus have been major pests of the nursery been routinely isolated around the world. Isolating industry in the PaciÞc Northwest (PNW) for nearly entomopathogens from soil provides insight into the 100 yr (Warner and Negley 1976). The major problem naturally occurring pathogen biodiversity and pro- species are black vine weevil and the root vides a pool of potential biological control agents. weevil, Otiorhynchus ovatus L. (Coleoptera: Curcu- Surveys of entomopathogen diversity commonly focus lionidae). Current black vine weevil management pro- on a single pathogen group (entomopathogenic fungi, grams center around the use of broad spectrum in- nematodes, or bacteria). However, all of these groups secticide sprays targeted against adults. However, can occur simultaneously, each having their own po- entomopathogenic nematodes and fungi have also tential impact on insect populations. been shown to be effective control agents of black The black vine weevil, Otiorhynchus sulcatus F. vine weevil (Moorhouse et al. 1993a-c, 1994, Berry et (Coleoptera: Curculionidae), is a severe pest of Þeld al. 1997, Willmott et al. 2002). and container-grown ornamentals as well as small fruit Naturally occurring entomopathogens in the PNW crops worldwide (Moorhouse et al. 1992). The black are poorly characterized. The natural distribution of vine weevil is a polyphagous and univoltine insect. entomopathogenic nematodes in Oregon has been studied (Liu and Berry 1995, 1996), but no report has Mention of trades names or commercial products in this publication been made characterizing entomopathogenic fungi is solely for the purpose of providing speciÞc information and does not imply recommendation or endorsement by the U.S. Department of and bacteria occurrence in Oregon. There are no re- Agriculture. ports from Washington or Idaho characterizing ento- 1 Corresponding author; e-mail: [email protected]. mopathogen occurrence. The fungi of primary inter- 1336 ENVIRONMENTAL ENTOMOLOGY Vol. 33, no. 5 est in these studies were Beauveria bassiana (Balsamo) Pathogen Isolation. Entomopathogenic nematode Vuillemin (: ) and Metar- isolation was performed using the insect baiting hizium anisopliae (Metchnikoff) Sorokin (Hypocre- method (Bedding and Akhurst 1975). Five Galleria ales: Clavicipitaceae). M. anisopliae has been studied mellonella L. (Lepidoptera: Pyralidae) larvae (ob- for black vine weevil control in Europe (Poprawski et tained from Sunshine Mealworm, Silverton, OR) were al. 1985, Moorhouse et al. 1993a-c, 1994). B. bassiana placed on the soil surface of two petri dishes (15 by 100 is an ubiquitous with a broad host range and has mm) per sample. Petri dishes were nearly Þlled with been studied extensively for control of a wide variety soil, inverted, and incubated at 22ЊC. Larvae were of insect pests (Hajek and St. Leger 1994). Bacillus removed from the petri dishes after 7Ð10 d. The assay thuringiensis Berliner (Bacillales: Bacillaceae) has a was repeated using fresh G. mellonella in the same soil long history as an effective biological control agent of sample for another 7Ð10 d. Soil was kept moist (ap- a number of insect pests in several orders, but none of proximately Þeld capacity) during isolation. Ento- the strains tested to date are particularly effective mopathogenic nematodes were isolated from G. mel- against Otiorhynchus spp. (Anonymous 2002). While lonella cadavers showing signs of infection (Kaya and there are several nematode species currently being Stock 1997). Larvae infected with nematodes were sold for black vine weevil larval control, strains with placed individually on White traps (White 1927) and increased efÞcacy, persistence, and a more favorable held at 22ЊC, and infective juveniles (IJ) were col- range of operating temperatures are needed. lected and stored at 7ЊC. The entomopathogenic nem- The objectives of these studies were to characterize atodes collected were exposed to fresh G. mellonella nematode, fungal, and bacterial entomopathogen oc- larvae to conÞrm pathogenicity. Nematodes were currence in Þeld-grown nursery soils from the major identiÞed by Dr. Byron Adams (Department of En- nursery production areas in Oregon, Washington, and tomology and Nematology, University of Florida). Idaho, as well as determine their virulence against Fungal isolation was performed using insect baiting black vine weevil. (Zimmermann 1986) as described above. Larvae in- fected with fungi were placed individually in snap- lock petri dishes (9 by 50 mm; Becton Dickinson, Materials and Methods Franklin Lakes, NJ) with a piece of moist Þlter paper Soil Sampling. Soil samples were collected JulyÐ (Whatman no. 1). Infected larvae were held at 22ЊC September 2002 from commercial wholesale nursery and allowed to sporulate. In addition, semiselective Þelds in Oregon (20), Washington (7), and Idaho (8). media was used to isolate B. bassiana and M. anisopliae Sample collection in each state took place in the major from each soil sample. Brießy, 10 g of soil from each nursery production areas (OregonÑBenton, Clacka- sample were placed into 90 ml of a sterile 0.1% Tween mas, Lane, Marion, Polk, and Yamhill Counties; Wash- 80 solution, shaken for 20 min at 200 rpm on a rotary ingtonÑCowlitz, King, Pierce, Snohomish, and Thur- shaker, and sonicated for 2 min. One hundred micro- ston Counties; IdahoÑCanyon, Elmore, Gem, and liters of the suspension was spread onto media plates Payette Counties). Nurseries surveyed meet two basic selective for B. bassiana (Doberski and Tribe 1980) criteria: (1) they were located in the major nursery and M. anisopliae (Veen and Ferron 1966). Plates were production areas in each state and (2) black vine incubated at 28ЊC for 10 d. The fungi collected were weevil was known to occur in the area sampled. The identiÞed based on macro- and microscopic charac- production practices at each nursery were unique to teristics (Humber 1997). Individual colonies of the each grower, Þeld, and plant species, but all were respective fungi were removed from the selective me- typical of plant production practices throughout the dia and cultured on potato dextrose agar (PDA). industry. Plants in most nurseries sampled are har- After no more than two passes on PDA, single vested every 3Ð5 yr. A total of 280 samples were col- cultures of all fungal isolates were made, and each lected in all (Oregon, 170; Washington, 50; Idaho, 60). isolate was placed into long-term storage and cata- Samples were collected from a wide variety of Þeld- loged. Fungi were prepared for long-term storage by grown nursery stock. An individual sample consisted growing each isolate on PDA plates (15 by 100 mm) of 10 soil cores (2 cm wide by 25 cm deep) taken at 28ЊC until sporulation. Using sterile technique, a randomly over an area of Ϸ20 m2 that contained the plate was ßooded with 10 ml of cryo-preservation same plant species. Because all samples were collected solution (0.5% Tween 80 and 20% glycerol), and the from commercial wholesale nurseries, Þeld moisture surface of the plate was gently agitated with a sterile throughout the growing season was maintained near loop to place the into suspension. One milliliter Þeld capacity to maximize plant growth. The 10 cores of the conidial suspension was pipetted into cryo-vials making up each sample were combined into a large (Nalgene Co., Rochester, NY). Once three vials were (12 by 25 cm) sterile plastic bag (Nasco, Modesto, Þlled for each isolate, they were mixed with a vortex CA), mixed thoroughly, and placed in a cooler on ice genie and placed directly into a freezer at Ϫ80ЊC. until returned to the laboratory. Five to 10 samples The selective process for isolation of B. thuringiensis were collected from each nursery. Soil samples from from soil was adapted from Travers et al. (1987) and each location represented all areas of the nursery. The Martin and Travers (1989), along with the modiÞca- soil corer was disinfected with 95% ethanol between tions of Morris et al. (1998). Brießy,1gofsoil was samples. Samples were stored up to7dat4ЊC until added to 20 ml of L-broth (tryptone 10 g, yeast extract processed. 5g,andNaCl5gin1liter of distilled water, pH 6.8), October 2004 BRUCK:ENTOMOPATHOGENS IN PACIFIC NORTHWEST NURSERY SOILS 1337 supplemented with sodium acetate buffer (0.25 M, pH heart Cup Co., Owings Mills, MD) with artiÞcial diet 6.8), in a 125-ml ßask and shaken (250 rpm, 30ЊC) for (Shanks and Finnigan 1973) and incubated at 22ЊC for 4 h. Samples of 500 ␮l were heat-treated at 80ЊC for 3 14 d, and larval mortality was recorded. Assays were min. One hundred Þfty microliters of each culture was performed at 22ЊC to simulate typical soil tempera- spread onto LBA plates (tryptone 10 g, yeast extract tures when last-instar black vine weevils are present in 5g,andNaCl5gin1liter of distilled water, pH 6.8, the Þeld. A germination test of each isolate was per- agar 15 g/liter, three plates/sample) and incubated at formed for both runs of the bioassay to ensure a high 30ЊC overnight. Using sterile toothpicks, 30 colonies level (90Ð100%) of viability (Goettel and Inglis 1997). were transferred onto three (10 colonies/plate) T3A All experiments included an untreated control (0.1% plates (tryptone 3g, tryptose 2 g, yeast extract 1.5 g, Tween 80) and were arranged in a completely ran- and MnCl2 0.005 g in 1 liter of 50 mM phosphate buffer, domized design. pH 6.8, agar 15 g/liter) and incubated for 40 h at 30ЊC. The ability of the entomopathogenic nematode col- All colonies with typical B. thuringiensis colony mor- lected to infect last-instar black vine weevil at 10, 12, phology (fried egg) from the T3A plates were exam- 15, and 22ЊC was determined. The nematode was ϫ ined using phase contrast at 1,000 for spores and reared on last-instar G. mellonella at 22ЊC (Kaya and crystals indicative of B. thuringiensis (Tanada and Stock 1997) and was not subcultured more than twice Kaya 1993). in G. mellonella before use in experiments. Ten last- Long-term storage of the B. thuringiensis was per- instar black vine weevil were held individually in snap- formed using 48-h pure cultures grown on nutrient lock petri plates (9 by 50 mm; Becton Dickinson) agar (NA). One milliliter of sterile nutrient broth containing Þlter paper (Whatman no. 1). An aliquot (NB) with 17% glycerol was pipetted into a cryo-vial. containing 400 IJ was added to each dish. Dishes were A loop full of B. thuringiensis from a single cell culture incubated at each temperature for 14 d, and larval was placed into the vial, mixed with a vortex genie, and Ϫ Њ mortality was recorded. The nematode bioassay was immediately placed on ice until frozen at 80 C. performed four times. All experiments included an Laboratory Bioassays. Bioassays against the black untreated control (water only) and were arranged in vine weevil of the prominent pathogens from each a completely randomized design. group collected were performed. Five isolates of B. All B. thuringiensis isolates collected were screened bassiana (OregonÑIP43, IP54, IP57, IP63, and IP237; for activity against black vine weevil adults. Isolates WashingtonÑIP113, IP135, IP325, IP350, and IP351; were taken from long-term storage and grown for 24 h and IdahoÑIP370, IP372, IP375, IP376, and IP377) and on NA. Bafßed ßasks (250 ml) containing 100 ml of NB M. anisopliae (OregonÑIP37, IP38, IP39, IP272, and were inoculated with a loopful of 24-h cultures of B. IP276; WashingtonÑIP45, IP99, IP131, IP285, and thuringiensis and incubated on a rotary shaker (250 IP326; and IdahoÑIP79, IP80, IP86, IP368, and IP515) rpm, 30ЊC) for 5Ð6 d until a majority of the cells had from each state were randomly selected and screened lysed. The culture was centrifuged at 10,000 rpm for 5 against last-instar black vine weevil. Each of the fungal Њ isolates assayed originated from an individual soil sam- min at 15 C, decanted, and resuspended in 100 ml ple collected from different nurseries. Isolates were dH2O. The suspension was centrifuged as above, de- selected in this way in an attempt to maintain their canted, and resuspended in 25 ml dH2O. With the use genetic uniqueness, because most nurseries were sep- of a hemocytometer, the spore concentration was ad- ϫ 7 arated by several kilometers. Black vine weevil larvae justed to 1 10 spores/ml. It is recommended when were obtained from a laboratory colony maintained at screening Lepidoptera with unknown B. thuringiensis the USDA-ARS Horticultural Crops Research Labo- isolates to do so with a mixed culture of spores and ratory, Corvallis, OR. The isolates used in the bioassay crystals (McGuire et al. 1997). The same approach has were taken from pure cultures in long-term storage, been taken here for screening unknown B. thuringien- grown on PDA at 28ЊC, and allowed to sporulate. Two sis isolates for activity against black vine weevil. The plates (15 by 100 mm) were ßooded with 10 ml of a bioassay was performed by spraying the suspension of sterile 0.1% Tween 80 solution, and the spores were each isolate onto one strawberry leaf until runoff. The removed by gentle agitation with a sterile loop. He- treated leaves were placed in a laminar ßow hood and mocytometer counts of all suspensions were made, allowed to dry. Once dry, the leaf petioles were placed and the spore concentration was adjusted to 1 ϫ 104 into an aqua tube (Syndicate Sales, Kokomo, IN) Þlled spores/ml. For the Þrst run of the bioassay, a total of with water to maintain freshness. A single leaf was 20 last-instar black vine weevil were assayed for each placed into a large plastic deli container (C18Ð5032; isolate. Because of a limited number of , 10 Pactiv, Lake Forest, IL) along with Þve black vine larvae were used per isolate on the second run of the weevil adults. Each treatment was replicated four bioassay. The bioassays were performed on separate times. The containers were held at 22ЊC and a 18:6 days, and fresh spore suspensions were made for the (L:D) cycle for 3d. After 3d, treated leaves were second run of the bioassay. Assays of M. anisopliae and removed and replaced with untreated strawberry B. bassiana assays were performed separately. Larvae leaves. The weevils were allowed to continue feeding were submerged, in groups of Þve, into 5 ml of spore on untreated leaves for 7Ð10 d. At the conclusion of the suspension (used only once) for 1 min and placed on experiment, all dead weevils were examined micro- Þlter paper to remove excess solution. Larvae were scopically (1,000ϫ) to determine if they were infected then placed individually into 1-oz plastic cups (Sweet- with B. thuringiensis. All experiments included a water 1338 ENVIRONMENTAL ENTOMOLOGY Vol. 33, no. 5

Table 1. Location of wholesale nurseries sampled and pathogens isolated

Metarhizium Beauveria Paecilomyces Bacillus Steinernema Location Sand:Silt:Clay anisopliae bassiana tenuipes thuringiensis oregonense Caldwell, ID (1)a 33:45:22 ߛ Caldwell, ID (2) 18:55:27 ߛߛ Emmet, ID (1) 17:51:32 ߛߛ Emmet, ID (2) 32:41:27 ߛߛ Idaho City, ID 38:42:20 ߛߛߛ Meridian, ID (1) 60:30:10 ߛߛ Meridian, ID (2) 26:50:24 Nampa, ID 23:62:15 ߛߛ Aurora, OR (1) 34:49:17 ߛߛ Aurora, OR (2) 31:55:14 ߛߛ Aurora, OR (3) 26:55:19 ߛߛ Aurora, OR (4) 71:25:4 ߛ Aurora, OR (5) 21:57:22 ߛߛ Canby, OR 44:40:16 Corvallis, OR 44:34:22 ߛ Gaston, OR 11:55:34 ߛ Hubbard, OR 9:57:34 ߛߛ Independence, OR (1) 11:53:36 ߛߛ Independence, OR (2) 46:33:21 ߛߛ ߛ Lowell, OR 38:37:25 ߛߛ McMinnville, OR 17:52:31 ߛ Monmouth, OR 46:28:26 ߛ Mt. Angel, OR 7:58:35 ߛߛ Salem, OR 72:14:14 ߛ Silverton, OR 24:32:44 ߛ St. Paul, OR 29:44:27 Stayton, OR 49:29:22 ߛߛ Woodburn, OR 18:52:30 ߛ Burien, WA 39:45:16 ߛߛ Des Moines, WA 24:56:20 ߛߛ Kirkland, WA 67:16:17 ߛ Longview, WA 35:43:22 ߛߛ Olympia, WA 51:36:14 ߛߛߛ ߛ Puyallup, WA 55:33:12 ߛ Seattle, WA 48:36:16 ߛߛߛ

a Notes multiple nurseries located in same city. control and were arranged in a completely random- entomopathogenic nematodes and B. thuringiensis ized design. were lower than has been found in other areas Data Analysis. Because of a lack of infection in the (Akhurst and Brooks 1984, Martin and Travers 1989, nematode and B. thuringiensis assays, no analysis were Hara et al. 1991, Liu and Berry 1995, Shapiro-Ilan et al. performed on these data. Data from the fungal bioas- 2003). says were nonparametric and were analyzed using a ␹2 The frequency of nematode isolations from this analysis (SAS Institute 1999). Data from the two bio- survey was Ͻ1% (2/280). Steinernema oregonense assays were considered replicates, and a ␹2 analysis (Rhabditidae: Steinernematidae) was collected from was performed on the combined data for each fungus two soil samples from a single nursery in Olympia, WA. to determine if there were any signiÞcant differences S. oregonense was originally isolated from the in larval mortality between isolates. A 2 by 2 Fisher Oregon coast, and until now, had not been isolated exact test was used to determine if mortality differ- from noncoastal areas (Liu and Berry 1995). Only ences between isolates of the same fungus were sig- considering S. oregonense occurrence in Washington, niÞcantly different (SAS Institute 1999). Because of nematode frequency was still low at 4% (2/50). The the large number of treatments in the nonparametric insect bait method has been successfully used (Bed- test, a reference probability of P Յ 0.01 was used ding and Akhurst 1975, Liu and Berry 1995, Shapiro- throughout the data analysis. Ilan et al. 2003) for isolating entomopathogenic nem- atodes from soil. In this study, only nematodes infective to G. mellonella were isolated; thus, other Results and Discussion species or strains of nematodes may have gone unde- Soil-borne entomopathogens occur widely tected. For example, Steinernema scapterisci Nguyen throughout the PNW (Table 1). Entomopathogenic and Smart (Rhabditidae: Steinernematidae) is quite fungi were the most commonly isolated pathogen speciÞc to Scapteriscus spp. (Orthoptera: Gryllotalpi- group, and their occurrence was typical of collections dae) (Nguyen and Smart 1990). Direct extraction of elsewhere (Harrison and Gardner 1991, Bing and nematodes was not performed in this study and may Lewis 1993, Chandler et al. 1997, Klingen et al. 2002, have resulted in additional isolations. Also, had the Shapiro-Ilan et al. 2003). However, the occurrence of insect baiting been performed at higher temperatures October 2004 BRUCK:ENTOMOPATHOGENS IN PACIFIC NORTHWEST NURSERY SOILS 1339

Table 2. Occurrence of entomopathogenic fungi, nematodes, and bacteria collected from Pacific Northwest nursery soils

State Total meana Oregon Washington Idaho Number of nurseries sampled 20 7 8 35 Number of soil samples 170 50 60 280 Number of nurseries with M. anisopliae 15 7 7 29 Percent soil samples with M. anisopliae 21 72 35 34 Number of nurseries with B. bassiana 11 5 6 22 Percent soil samples with B. bassiana 16 22 20 18 Number of nurseries with P. tenuipes 1215 Percent soil samples with P. tenuipes 314 2 5 Number of nurseries with nematodes 0 1 0 1 Percent soil samples with nematodes 0 4 0 0.7 Number of nurseries with B. thuringiensis 2002 Percent soil samples with B. thuringiensis 4 0 0 0.7

a Total number of nurseries or samples collected or the mean percentage of samples collected with each pathogen.

(25ЊC), additional nematode isolations may have been if there were any signiÞcant relationships between soil made. Therefore, these results should be considered as composition and nematode abundance. The soil com- a conservative estimate of entomopathogenic nema- position of one sample from each location surveyed is tode occurrence. presented for comparison (Table 1). While Heterorhabditis and Steinernema spp. have The strain of S. oregonense that was collected in- previously been found to be widely distributed in fected only a single black vine weevil larvae at 15 and Oregon, with the highest occurrence in costal soils, 22ЊC over the four runs of the bioassay. Based on these neither was found to occur in the Willamette Valley data, this particular strain of S. oregonense would not (the center of nursery production in Oregon and the be a good candidate for further development as a location of samples in this study) (Liu and Berry biological control agent for black vine weevil, partic- 1995). The impact of agricultural activities such as ularly for use in the PNW where cool wet conditions tillage and the application of chemical pesticides and prevail in the fall and spring months, when nematodes fertilizers have been found to have mixed results in are applied for black vine weevil control. relationship to nematode survival and persistence. Or- Metarhizium anisopliae was isolated most fre- namental nursery plants are grown in intensively man- quently, followed by B. bassiana and Paecilomyces aged agricultural systems generally consisting of high tenuipes (Peck) Samson (Eurotiales: Trichoco- levels of fertilizer and pesticide use. Organophosphate maceae) (Table 2). The occurrence of M. anisopliae in nematicides such as Mocap (ethoprop) is routinely nursery soils in Washington and Idaho was particularly used in the ornamental nursery industry. Broad spec- common, with at least one soil sample positive from trum biocides (methyl bromide and Vapam) are also every nursery surveyed in Washington and seven of used before planting by some growers. The long-term eight nurseries in Idaho. B. bassiana was also widely use of chemical fertilizers and pesticides in nursery distributed. P. tenuipes was not as common, occurring production may be a potential cause for the lack of in only one nursery in Oregon, three in Washington, entomopathogenic nematodes. Tillage does not have and one in Idaho. The use of both insect baiting and a signiÞcant impact on the survival of Heterorhabditis semiselective media proved important to obtain the bacteriophora (Rhabditidae: Heterorhabditidae), has most accurate estimate of entomopathogenic fungi a positive impact on Steinernema riobrave (Rhabditi- occurrence in the soil. This was particularly true for M. dae: Steinernematidae), and has a negative impact on anisopliae (Table 3). Steinernema carpocapsae (Rhabditidae: Steinernema- It has been proposed that habitat, not insect selec- tidae) (Millar and Barbercheck 2002). Entomopatho- tion, drives the population structure of M. anisopliae genic nematode persistence is enhanced in conserva- and B. bassiana (Bidochka et al. 1998, 2002). M. aniso- tion tillage systems with increased levels of crop pliae occurs more frequently in agricultural habitats, residue (Shapiro et al. 1999, Hummel et al. 2002). Even though ornamental nursery plants are perennially Table 3. Percent isolation of Metarhizium anisopliae and grown, intense tillage of the soil between plant rows Beauveria bassiana via Galleria baitinga and semi-selective mediab is common. Prolonged exposure to inorganic fertiliz- ers inhibit nematode infectivity, while short-term ex- Metarhizium anisopliae Beauveria bassiana posure generally is not detrimental (Bednarek and Insect Baiting 23% 17% Gaugler 1997). Soil texture also effects nematode sur- Selective Media 11% 1% vival, dispersal, and persistence. As clay content in- Totalc 34% 18% creases, nematode survival (Kung et al. 1990) and dispersal (Georgis and Poinar 1983, Barbercheck and a (Bedding and Akhurst 1975, Zimmermann 1986). b Metarhizium anisopliae (Veen and Ferron 1966); Beauveria bas- Kaya 1991) decrease. Because of the extremely low siana (Doberski and Tribe 1980). frequency of nematode isolation, it was not possible to c Percent of the total samples collected from all three states positive perform meaningful correlation analyses to determine for M. anisopliae (34%; 95/280) and B. bassiana (18%; 50/280). 1340 ENVIRONMENTAL ENTOMOLOGY Vol. 33, no. 5

Fig. 1. Mean mortality from the bioassay (N ϭ 30) of last-instar O. sulcatus after 14-d exposure to M. anisopliae isolates (1 ϫ 104 spores/ml) collected from PNW nursery soils. Isolate means that share the same letter are not signiÞcantly different as determined by Fisher exact test (SAS Institute 1999). whereas B. bassiana is more frequently isolated from IP79, IP99, and IP285 all caused at least 53% mortality, forested habitats (Bidochka et al. 1998). While the with IP99 causing 73% mortality (Fig. 1). All of the B. number of nurseries with M. anisopliae and B. bassiana bassiana isolates tested caused infection (based on are similar (Table 2), there were nearly twice as many macro- and microscopic characteristics), but only iso- individual soil samples positive for M. anisopliae (95) lates IP113, IP135, IP370, IP372, IP377, and IP375 as there were B. bassiana (50). The occurrence of B. caused signiÞcantly higher mortality than the control bassiana and M. anisopliae have been shown to be (P Յ 0.01; Fig. 2). Wide variability in weevil larval negatively correlated with tillage intensity. Conserva- mortality between Þeld-collected isolates of the same tion and no-till systems are most compatible for the fungus has also been observed with the pecan weevil survival of soil-borne B. bassiana and M. anisopliae (Shapiro-Ilan et al. 2003). The most virulent isolates (Bing and Lewis 1993, Hummel et al. 2002). Pesticide have been deposited in the USDA-ARS Collection of usage (Hummel et al. 2002) and fresh cow manure Entomopathogenic Fungal Cultures, Ithaca, NY. (Rosin et al. 1996) also reduce entomopathogenic Bacillus thuringiensis was isolated from Oregon, but fungal survival and persistence. B. bassiana and M. not Washington or Idaho soils (Tables 1 and 2). B. anisopliae occurrence overall in soils collected from thuringiensis has a worldwide distribution and has pecan orchards has been shown to be negatively cor- been isolated from soils in a number of different en- related with manganese levels, whereas calcium and vironments (Martin and Travers 1989). While the magnesium levels are positively correlated with M. presence of insects does not have any noticeable effect anisopliae occurrence (Shapiro-Ilan et al. 2003). Be- on B. thuringiensis occurrence, there are some areas cause individual soil samples were not analyzed in this that are more rich in B. thuringiensis than others study, it is unknown if a similar relationship occurs (Bernhard et al. 1997). Eastern Asian soils are partic- between micronutrients. The amounts of sand, silt, or ularly high (78% of soil samples) in B. thuringiensis, clay in the single soil sample characterized from each whereas B. thuringiensis made up only 25% of the location and the occurrence of M. anisopliae or B. colonies examined in soils from the United States bassiana at that location were not signiÞcantly corre- (Martin and Travers 1989). In agricultural Þelds in lated (SAS Institute 1999). particular, the frequency of B. thuringiensis in the Fungal bioassays revealed a wide range of virulence United States was only one-half that of the Republic between isolates. All M. anisopliae isolates assayed but of Korea (Martin and Travers 1989). The reason why three (IP38, IP39, and IP270) caused substantially B. thuringiensis was not isolated from nursery soils in higher larval mortality than was found in the control Washington and Idaho is unclear. To test my isolation (P Յ 0.01; Fig. 1). The M. anisopliae isolates IP37, IP45, technique, a known culture of B. thuringiensis (HD-1) October 2004 BRUCK:ENTOMOPATHOGENS IN PACIFIC NORTHWEST NURSERY SOILS 1341

Fig. 2. Mean mortality from the bioassay (N ϭ 30) of last-instar O. sulcatus after 14-d exposure to B. bassiana isolates (1 ϫ 104 spores/ml) collected from PNW nursery soils. Isolate means that share the same letter are not signiÞcantly different as determined by Fisher exact test (SAS Institute 1999). was subjected to the selection procedure, and all 30 Acknowledgments colonies examined on the T3A plates were B. thurin- I thank all of the nursery growers that allowed collection giensis. None of the B. thuringiensis isolates collected of soil samples; A. GrifÞth, K. Donahue, and A. Spycher for and screened were found to be pathogenic to black assistance in collecting soil samples, isolating entomopatho- vine weevil adults. Because of the complete lack of gens, and performing bioassays; B. Adams for nematode iden- infectivity and a limited supply of adult weevils, the B. tiÞcation; and J. Spatafora for assistance with fungal identi- thuringiensis bioassay was not repeated. Þcation. This work was supported by a grant from the USDAÐ ARS, Northwest Center for Nursery Crop Research. Overall, the occurrence of entomopathogenic fungi from PNW nursery soils was typical of results from other surveys performed throughout the world. The occurrence of entomopathogenic nematodes and B. References Cited thuringiensis was less than was observed in other stud- Akhurst, R. J., and W. M. Brooks. 1984. The distribution of ies. The reason for the lack of nematode and bacteria entomophilic nematodes (Heterorhabditidae and Stein- isolations is unknown. The implementation of multiple ernematidae) in North Carolina. J. Invertebr. Pathol. 44: isolation techniques may have resulted in the collec- 140Ð145. tion of additional nematode and bacteria isolates, as Anonymous. 2002. SpeciÞcity database. http://www.glfc. was the case for the fungi. It is also possible that the forestry.ca/bacillus/. detection techniques for each pathogen group are not Barbercheck, M., and H. K. Kaya. 1991. Effect of host con- dition and soil texture on host Þnding by the entomog- equal. For instance, had the insect baiting for ento- enous nematodes Heterhabditis bacteriophora (Rhab- mopathogenic nematodes been performed at higher ditida: Heterorhabditidae) and Steinernema carpocapsae temperatures, additional isolations may have been (Rhabditida: Steinernematidae). Environ. Entomol. 20: made. In addition, soil samples in this study were 582Ð589. collected only during the summer months, and the Bedding, R. A., and R. J. Akhurst. 1975. A simple technique occurrence of entomopathogens in the soil may vary for the detection of insect parasitic rhabditid nematodes temporally throughout the year. Future studies will in soil. Nematologica. 21: 582Ð589. concentrate on determining the ability of the most Bednarek, A., and R. Gaugler. 1997. Compatability of soil amendments with entomopathogenic nematodes. J. efÞcacious fungal isolates (IP37, IP45, IP79, IP99, and Nematol. 29: 220Ð227. IP285) collected in these studies, as well as potentially Bernhard, K., P. Jarrett, M. Meadows, J. Butt, D. J. Ellis, G. M. other yet to be determined isolates, to control black Roberts, S. Pauli, P. Rodgers, and H. D. Burges. 1997. vine weevil. Natural isolates of Bacillus thuringiensis: worldwide dis- 1342 ENVIRONMENTAL ENTOMOLOGY Vol. 33, no. 5

tribution, characterization and activity against insect dae and Steinernematidae) in Oregon soils. Environ. En- pests. J. Invertebr. Pathol. 70: 59Ð68. tomol. 24: 159Ð163. Berry, R. E., J. Liu, and E. Groth. 1997. EfÞcacy and per- Liu, J., and R. E. Berry. 1996. Steinernema orgonensis n. sp. sistence of Heterorhabditis marelatus (Rhabditida: Het- (Rhabditida: Steinernematidae) from Oregon, U.S.A. erorhabditidae) against root weevils (Coleoptera: Cur- Fundam. Appl. Nematol. 19: 375Ð380. culionidae) in strawberry. Environ. Entomol. 26: 465Ð Martin, P.A.W., and R. S. Travers. 1989. Worldwide abun- 470. dance and distribution of Bacillus thuringiensis isolates. Bidochka, M. J., J. E. Kasperski, and G.A.M. Wild. 1998. Appl. Environ. Microbiol. 55: 2437Ð2442. Occurrence of the entomopathogenic fungi Metarhizium McGuire, M. R., L. J. Galan-Wong, and P. Tamez-Guerra. anisopliae and Beauveria bassiana in soils from temperate 1997. Bacteria: bioassay of Bacillus thuringiensis against and near-northern habitats. Can. J. Bot. 76: 1198Ð1204. Lepidopteran Larvae, pp. 91Ð99. In L. A. Lacey (ed.), Bidochka, M. J., F. V. Menzies, and A. M. Kamp. 2002. Ge- Manual of techniques in insect pathology. Academic, San netic groups of the insect-pathogenic fungus Beauveria Diego, CA. bassiana are associated with habitat and thermal growth Millar, L. C., and M. E. Barbercheck. 2002. Effects of tillage preferences. Arch. Microbiol. 178: 531Ð537. practices on entomopathogenic nematodes in a corn Bing, L. A., and L. C. Lewis. 1993. Occurrence of the en- agroecosystem. Biol. Control. 25: 1Ð11. tomopathogen Beauveria bassiana (Balsamo) Vuillemin Moorhouse, E. R., A. K. Charnely, and A. T. Gillespie. 1992. in different tillage regimes and in Zea mays L. and viru- Review of the biology and control of the vine weevil, lence towards Ostrinia nubilalis (Hu¨ bner). Agric. Eco- Otiorhynchus sulcatus (Coleoptera: Curculionidae). Ann. syst. Environ. 45: 147Ð156. Appl. Biol. 121: 431Ð454. Chandler, D., D. Hay, and A. P. Reid. 1997. Sampling and Moorhouse, E. R., A. T. Gillespie, and A. K. Charnley. 1993a. occurrence of entomopathogenic fungi and nematodes in Application of Metarhizium anisopliae (Metsch.) Sor. UK soils. Appl. Soil Ecol. 5: 133Ð141. conidia to control Otiorhynchus sulcatus (F.) (Co- Doberski, J. W., and H. T. Tribe. 1980. Isolation of ento- leoptera: Curculionidae) larvae on glasshouse pot plants. mogenous fungi from elm bark and soil with reference to Ann. Appl. Biol. 122: 623Ð636. ecology of Beauveria bassiana and Metarhizium anisop- Moorhouse, E. R., M. A. Easterbrook, A. T. Gillespie, and liae. Trans. Br. Mycol. Soc. 74: 95Ð100. A. K. Charnley. 1993b. Control of Otiorhynchus sulcatus Georgis, R., and G. O. Poinar Jr. 1983. Effect of soil texture (Fabricius) (Coleoptera: Curculionidae) larvae on a on the distribution and infectivity of Neoaplectana glaseri range of hardy ornamental nursery stock species using the (Nematode:Steinernematidae). J. Nematol. 15: 329Ð332. entomogenous fungus Metarhizium anisopliae. Biocontrol Goettel, M. S., and G. D. Inglis. 1997. Fungi: , Sci. Tech. 1: 63Ð72. pp. 211Ð249. In L. A. Lacey (ed.), Manual of techniques Moorhouse, E. R., A. T. Gillespie, and A. K. Charnley. 1993c. in insect pathology. Academic, San Diego, CA. The development of Otiorhynchus sulcatus (Fabricius) Hajek, A. E., and R. J. St. Leger. 1994. Interactions between (Coleoptera: Curculionidae) larvae on a range of orna- fungal pathogens and insect hosts. Annu. Rev. Entomol. mental pot-plant species and the potential for control 39: 293Ð322. using Metarhizium anisopliae. J. Hortic. Sci. 68: 627Ð635. Hara, A. H., R. Gaugler, H. K. Kaya, and L. M. Lebeck. 1991. Moorhouse, E. R., A. T. Gillespie, and A. K. Charnley. 1994. Natural populations of the entomopathogenic nematodes The inßuence of temperature on the susceptibility of vine (Rhabditida: Heterorhabditidae, Steinernematidae) weevil, Otiorhynchus sulcatus (Fabricius) (Coleoptera: from the Hawaiian Islands. Environ. Entomol. 20: 211Ð Curculionidae), larvae to Metarhizium anisopliae (Deu- 216. teromycotina: Hyphomycetes). Ann. Appl. Biol. 124: 185Ð Harrison, R. D., and W. A. Gardner. 1991. Occurrence of 193. entomogenous fungus Beauveria bassiana in pecan or- Morris, O. N., V. Converse, P. Kanagaratnam, and J.-C. Cote´. chard soils in Georgia. J. Entomol. Sci. 26: 360Ð366. 1998. Isolation, characterization, and culture of Bacillus Homminick, W. M. 2002. Biogeography, pp. 115Ð143. In R. thuringiensis from soil and dust from grain storage bins Gaugler (ed.), Entomopathogenic nematology. CABI and their toxicity for Mamestra configurata (Lepidoptera: Publishing, New York. Noctuidae). Can. Entomol. 130: 515Ð537. Humber, R. A. 1997. Fungi: identiÞcation, pp. 153Ð185. In Nguyen, K. B., and G. C. Smart Jr. 1990. Steinernema scap- L. A. Lacey (ed.), Manual of techniques in insect pathol- terisci n. sp. (Rhabditida: Steinernematidae). J. Nematol. ogy. Academic, San Diego, CA. 22: 187Ð199. Hummel, R. L., J. F. Walgenbach, M. E. Barbercheck, G. G. Poprawski, T. J., M. Marchal, and P.-H. Robert. 1985. Com- Kennedy, G. D. Hoyt, and C. Arellano. 2002. Effects of parative susceptibility of Otiorhynchus sulcatus and Sitona production practices on soil-borne entomopathogens in lineatus (Coleoptera: Curculionidae) early stages to Þve western North Carolina vegetable systems. Environ. En- entomopathogenic Hyphomycetes. Environ. Entomol. tomol. 31: 84Ð91. 14: 247Ð253. Kaya, H. K., and S. P. Stock. 1997. Techniques in insect Rosin, F., D. I. Shapiro, and L. C. Lewis. 1996. Effects of nematology, pp. 281Ð324. In L. A. Lacey (ed.), Manual of fertilizers on the survival of Beauveria bassiana. J. Inver- techniques in insect pathology. Academic, San Diego, tebr. Pathol. 68: 194Ð195. CA. SAS Institute. 1999. The SAS statistical system, version 8. Klingen, I., J. Eilenberg, and R. Meadow. 2002. Effects of SAS Institute, Cary, NC. farming system, Þeld margins and bait insect on the oc- Shanks, C. H., Jr., and B. Finnigan. 1973. An artiÞcial diet for currence of insect pathogenic fungi in soils. Agric. Eco- Otiorhynchus sulcatus larvae. Ann. Entomol. Soc. Am. 66: syst. Environ. 91: 191Ð198. 1164Ð1166. Kung, S.-P., R. Gaugler, and H. K. Kaya. 1990. Soil type and Shapiro, D. I., J. J. Obrycki, L. C. Lewis, and J. J. Jackson. entomopathogenic nematode persistence. J. Invertebr. 1999. Effects of crop residue on the persistence of Stein- Pathol. 55: 401Ð406. ernema carpocapsae. J. Nematol. 31: 517Ð519. Liu, J., and R. E. Berry. 1995. Natural distribution of ento- Shapiro-Ilan, D. I., W. A. Gardner, J. R. Fuxa, B. W. Wood, mopathogenic nematodes (Rhabditida: Heterorhabditi- K. B. Nguyen, B. J. Adams, R. A. Humber, and M. J. Hall. October 2004 BRUCK:ENTOMOPATHOGENS IN PACIFIC NORTHWEST NURSERY SOILS 1343

2003. Survey of entomopathogenic nematodes and fungi Warner, R. E., and F. B. Negley. 1976. The genus Otiorhyn- endemic to pecan orchards of the Southeastern United chus in America north of Mexico (Coleoptera: Curcu- States and their virulence to the pecan weevil (Co- lionidae). Proc. Entomol. Soc. Wash. 78: 240Ð262. leoptera: Curculionidae). Environ. Entomol. 32: 187Ð195. White, G. F. 1927. A method for obtaining infective nem- Smith, F. F. 1932. Biology and control of the black vine atode larvae from cultures. Science. 66: 302Ð303. weevil. U. S. Department of Agriculture Technical Bul- Willmott, D. M., A. J. Hart, S. J. Long, R. N. Edmondson, and letin 325. U.S. Department of Agriculture, Washington, P. N. Edmondson. 2002. Use of a cold-active ento- DC. mopathogenic nematode Steinernema kraussei to control Tanada, Y., and H. K. Kaya. 1993. Insect pathology. Aca- overwintering larvae of the black vine weevil Otiorhyn- demic, San Diego, CA. chus sulcatus (Coleoptera: Curculionidae) in outdoor Travers, R. S., P.A.W. Martin, and C. F. Reichelderfer. 1987. strawberry plants. Nematology. 4: 925Ð932. Selective process for efÞcient isolation of Bacillus spp. Zimmermann, G. 1986. The “Galleria bait method” for de- Appl. Environ. Microbiol. 53: 1263Ð1266. tection of entomopathogenic fungi in soil. J. Appl. Ento- Veen, K. H., and P. Ferron. 1966. A selective medium for the mol. 102: 213Ð215. isolation of Beauveria tenella and of Metarhizium anisop- liae. J. Invertebr. Pathol. 8: 268Ð269. Received 7 November 2003; accepted 4 June 2004.