ENTOMOLOGY L.A. Lacey et al. (2007) Phytoparasitica 35(5):479-489

Gut Bacteria Associated with the Pacific Coast Wireworm, canus, Inferred from 16s rDNA Sequences and Their Implications for Control

L.A. Lacey, T.R. Unruh, H. Simkins and K. Thomsen-Archer

A multitude of bacteria have been isolated from the guts of several species. Some of these have been modified to interfere with the development of the host insect or with the development and transmission of plant and pathogens transmitted by the host insect. We surveyed the gut flora of the Pacific Coast wireworm. LeConte. a serious pest of potato, at two sites in Oregon and Washington. Isolates were obtained from surface-sterilized triturated larvae by dilution plating on standard media. A rich diversity of species was found in 86 isolates, including spore-formers, non-spore-formers and aerobic and facultatively anaerobic species collected on four sampling dates at each location. Twenty-one of the isolates were identified to species based on rDNA sequence (nine distinct species). An additional 34 isolates were identified to genus from the sequence data while six isolates could be assigned only to family based on sequence comparisons. Twenty-seven additional isolates were identified to species (9). genus (17) or family (1) based on side-by-side morphological comparisons with isolates identified from rDNA sequence. The most frequently isolated bacterium was Bacillus inegareriuni, followed by Rahnella aquarius. A naturally occurring bacterium found in the gut and/or environment of a targeted insect that is modified to express toxins or other detrimental substances could provide certain advantages (such as persistence and recycling) over inundatively applied microbial control agents, particularly within soil habitats. The hypothesis that these species or others from the survey represent candidates for genetic modification to provide control options for L. canus is discussed. KEY WORDS: Bacillus megateriuln; midgut flora: Rahnella aquatilis: spore formers: non- spore formers.

INTRODUCTION

A wide variety of bacteria are associated with , ranging from obligate endosym- biotes and pathogens. to facultative pathogens and others (9.24,3 1). These comprise a broad spectrum of anaerobic and aerobic spore forming and non-spore forming species including short- and long-term residents of the gut. The gut microbial flora of insects has been surveyed in several taxa of insects (7,9.25). Some bacteria isolated from pest species have been considered as candidates for genetic manipulation to create paratransgenic biological control agents (20,25.28). The Pacific coast wireworm. Li,nonius canus LeConte, is found in irrigated habitats in western North America, where it is a pest of grain and vegetable crops, especially potato tubers in the Pacific Northwest of the USA and elsewhere. In addition to tuber

Received May 30. 2007: accepted July 29, 2007: http://www.phyt0parasit1Ca.org posting Sept. 10. 2007. Yakima Agricultural Research Laboratory, USDA-ARS, Wapato, WA 98951. USA. Corresponding author [e- mail: [email protected]].

479 Phvtoparasitica 35:5, 2007 damage through tunneling and feeding, the presence of wireworm larvae in processed potatoes is highly undesirable. Control of wireworms traditionally relies on broad spectrum insecticides despite the low efficacy of this approach (18.26). Reduction of the use of broad spectrum insecticides will require development of alternative control strategies, such as microbial control agents, that are as efficacious and affordable as chemical pesticides. Microbial based insecticides have shown promise for some insect pests of potato (35) but their efficacy against wireworms has been limited. Engineering bacteria that are normally found in the midgut to become pathogenic for wireworm larvae may provide an opportunity for microbial control. With this ultimate objective, a survey of bacteria in L. canus was undertaken in the eastern part of Washington State and Oregon. MATERIALS AND METHODS Collection of wireworms Surveys were made in the summer of 2004 at two locations, the Hermiston Agricultural Research and Extension Center in Hermiston, Oregon (elevation 183 m) and the USDA Agricultural Experiment Research Farm near Moxee, Washington (elevation 472 m). Soil at the Hermiston site is an Adkins fine, sandy loam, pH 6.7-7.1. with organic matter 0.7%-1%. Soil at the Moxec site is Cleman very fine sandy loam, pH 7.4, with organic matter <1%. In late spring and early summer (May 5—June 28), wireworms were collected on four dates from the Hermiston site. Later in the summer

(July 19—Aug. 30), as wireworms became difficult to collect at the Hermiston site, L. canus larvae were collected on four dates from the Moxee site. Collections at the Moxee site were terminated after August 30, when wireworms became difficult to collect. Bait balls composed of one-third dry oatmeal and two-thirds soil held together with organdy netting were used as lures, as described previously by Horton and Landolt (14). Twenty bait balls were buried at a depth of 10-15 cm with a separation distance of 1-1.5 m for each collection date. Colored string tied to the baits facilitated retrieval. The baits were collected 3 to 4 d after placement and brought back to the laboratory for recovery of wireworm larvae. Isolation of enteric bacteria Whole larvae were used for isolating gut flora because of the difficulty of removing intact guts. Bacteria isolated from whole larvae were considered enteric rather than hemocoelic in origin for two reasons. Internal bacterial symbiotes, if any, would have required more complex media and handling to isolate. The non-spore- forming pathogens of insects have low pathogenicity, but once they gain entry into the hemocoel, multiply rapidly and cause death of the insect by septicemia (24,31). Only living active wireworms were used for isolations. Furthermore, no spore-forming pathogens are known from wireworms. The larvae were first surface-sterilized as described by Lacey and Brooks (21) by submersing them in 70% ethanol followed by dilute NaCIO (15% Clorox) and rinsing with sterile distilled water. Groups often randomly selected late instar larvae were then ground in 10 ml of sterile water using a tissue grinder (Pyrex model 7725- 19: Fisher Scientific. New Lawn, NJ, USA). A drop of Tween 80 (Fisher Scientific) was added to facilitate homogenization of the suspension. The stock suspension and 10and 10-2 dilutions of the suspension were plated onto Brain Heart Infusion A gar, Tryptic Soy Agar (Soybean-Casein Digest Agar) and Nutrient Agar (Hardy Diagnostics. Santa Maria, CA, USA). When available (on 4.v.04, l.vi.04, 21.vi.04, 19.vii.04728.vii.04, 18.viii.04), a second lot of ten larvae on each collection date was prepared in the same manner and subsequently pasteurized in sterile 15 ml screw top test tubes in an 80°C water bath for 12 min and then cooled rapidly in crushed ice. The pasteurized stock suspension was plated

480 L.A. Lacey et al. onto the three media as above. This procedure allowed selective isolation of spore-forming species. On the dates when fewer than 20 larvae were available, the 10 ml suspension with ten or fewer larvae was divided into two batches of 5 ml; then one of them was diluted and plated and the other was pasteurized and plated. When numbers of larvae retrieved from the field were fewer than ten (28.vi.04. 30.viii.04), they were all used for the isolation procedure without pasteurization. The plates were incubated at 25°C for 24- 72 h. depending on growth. On selected dates (19.vii. 28.vii. 18.viii. 30.viii), the stock solution was also plated on the three media under anaerobic conditions usin g gas packs (AnaeroGen Compact sachets: Fisher Scientific). Clean plates of the same medium upon which initial isolations were made were streaked with each resulting colony that exhibited a distinct morphology. Bacteria from each colony were observed microscopically before and after Gram staining. Representative distinct colonies for each date were then chosen for identification based on 16S rDNA sequence: additional colonies of like type were identified by morphological traits alone based on side-by-side comparisons with isolates identified by sequencing and the use of descriptions in Bergeys Manual (13). Characteristics used were colony color and appearance, cell morphologies, presence or absence of spores. and Gram staining. Each bacterial isolate was grown in nutrient broth (Dilco Amplification of 16S rDNA Laboratories, Detroit, Ml. USA) and the genomic DNA was extracted using a QIAGEN DNeasy Tissue Kit (QIAGEN Sciences, Germantown. MD, USA). The 16S rDNA of each isolate was amplified by PCR using universal eubacterial primers (Sigma Genosys. The ccgaattcgtcgacaacagagtttgatcCtggct.-3 and reverse Woodlands. TX, USA; forward 5- cttgttacgactt 3) which correspond to fD- 1 and rP-2 reported 5 cccgggatccaagcttacggctaC by Weisburg et al. (34) as suitable for most Eubacteria. The PCR was carried out in an MJ- 100 thermal cycler (Mi Research. Watertown, MA, USA) as follows: one cycle at 95°C for 2 mm, 30 cycles at 94°C for 30 s. 45°C for 30 s and 72°C for 2 mm, followed by 45°C for 1 min and 72°C for 2 mm. The PCR products were then purified with QlAquick PCR Purification Kits (QIAGEN Sciences) in preparation for cycle sequencing. Sequencing and identification Seven of the PCR products were sequenced commer- cially (Macrogen Inc., Seoul, Korea) and the remaining products were sequenced in house. Sequencing reactions consisted of 1 or 2 tl of the ABI, Big-dye v3.1 sequencing kit (Applied Biosystems. Foster City, CA, USA), with 3 pl of Big Dye sequencing buffer. I fil of template (50 ng PCR product) and I il (10 no) of primer with water to make 10 Pl. The reactions were carried out in a MJ-100 thermal cycler under the following conditions: one cycle at 96°C, followed by 25 cycles at 96°C for 30 s, 50°C for 15 s and 60°C for 4 mm. The products were purified with Sephadex G-50 (Sigma Chemical Co., St. Louis, MO, USA) columns and the sequences visualized with an ABI Prism 310 Genetic Analyzer (Applied Biosystems). Approximately 500-600 bp from each end of the 1440 bp 16S molecule was produced and used for identification. The sequence data were edited to retain only high-quality sequences and were com- pared with the BLAST algorithm (2) to 16S rDNA sequences as compiled and implemented in BIBI (Bioinformatics Bacterial Identification Tool; 8). Searches employed both ends of the 16S molecule when available; bacterial species were identified based on their similarity with existing sequences using both type and non-type isolates compiled in BIBI. Criteria used to accept sequence-based identifications were the position of the query sequence in

481 PhvtoparasitiCa 35:5. 2007 the neighbor joining (NJ) tree created in BIBI (parameters included pair-wise gap deletion, tree optimization with tree-cleaner). All identifications began using the nomenclature- compliant (stringent) 16S bacterial library in BIBI. If this search led to a NJ tree containing a discrete dade containing both type strains and uniformly a single species of the non- type strains, then this identification was accepted. In cases of poorly defined clades the nomenclature-stringent 16S type strain bacterial library was searched and a species ID was accepted only if the query fell into a distinct dade with two or more types. When multiple species but only one genus were observed in a well defined dade, then only the genus was reported.

RESULTS

Surveys for enteric bacteria of L. canus larvae from the vicinity of Hermiston, Oregon and Moxee. Washington yielded a rich diversity of species including spore-formers, non- spore-formers and aerobic and facultatively anaerobic species (Table 1). Eighteen of the 86 isolates were identified to species based on rDNA sequence: these isolates comprised nine distinct species. An additional 34 isolates were identified to genus from the sequence data: only seven isolates, all Enterobacteriaceae, could not be resolved below family from the sequence comparisons. The GenBank accession numbers for these sequences are provided in Table 1. Finally, 27 isolates were identified based on side-by-side morphological comparisons with isolates identified from rDNA sequence: nine to species. 17 to genus and one to family. The collected isolates include members from nine bacterial families and 15 genera (Table 2). Species in the Bacillaceae, Pseudomonadaceae and especially the Enterobac- teriaceae were abundantly represented. Diagnosis of the last family was most difficult and includes the seven isolates that could be resolved only to family. This may reflect closer relationships among species in the Enterobacteriaceae. None of the 17 isolates identified to the genus Pseudomonas could be resolved to species despite extremely high sequence similarities to various isolates in the genus. Like the Pseudomonadaceae, the five remaining families (Table 2) were each represented by a single genus. Bacillus (22 isolates, 16 to species. 6 to genus only) and Pseudomonas (23 isolates, all to genus only) were heavily represented and consisted of over half of the isolates. Bacillus megaterium was the most common of the bacterial species that were recovered from L. canus, occurring at both collection sites and on six of the eight collection dates including those at the very beginning and the end of our sampling. It was isolated on all three media, but grew most often (83% of the time) on Tryptic Soy Agar. Rahnella aquatilis was isolated on all four Moxee sampling dates and grew on all three media. The only two species that grew under anaerobic conditions were R. aquarius and Serratia narcescens.

482 L.A. Lacey et al.

TABLE i. Identifications of 86 isolates of bacteria from the guts of Limonius canus [Sample references, collection month and day in 2004 (e.g. 601A=June 1. 2004. sample A)] (1) Media Sample GenBank Identity Type and environmental isolates of Accession bacterial Species/Genera in dade with query used No. (BIBI) N 505-A DQ904569 Micrococcus iuteus2T 42. thailandicus I luteus 1T N 505-B DQ904570 Cellulosimicrobiuin ceUulans3T 20. t, 8, ce/lesea 2. sp. terreutfl I T 505-C DQ904571 Exiguobacterium sibiricu,n T 2. urtemiae I. undue 5. sp. antarcticu,fl 2. (,xidorolerans 4. acerihcu,n S T.B 505-F. DQ904572 Bacillus ,negaterium2T45 megaterium N.T.B 601-A DQ904573 Pseudomonas sp. Pseudomonas8T50 Pseudomonas OT50 N.B 601-B DQ904574 Pseudornonas sp. T 601-F DQ904575 Pseudomonas sp. Pseudomonas 41 50 Pseudonwna.c t4T N 601-F DQ904576 Pseudotnonas sp. 50 Leucobacter4T N 601-G 1 DQ904577 Leucobacter sp. T.B 621-A DQ904578 Pseudomonas sp. Pseudomonas l4T50 B 621-B DQ904579 Enterobacteriaceae Serratia, Rahnella. Yersinia T 621-D DQ904580 Pseudomonas Sf). PseUdOiflOnUS45T50 T 621-F DQ90458 I Bacillus megater,um2T49. flexus / megaterium Ex,guobacteriurn4T T 621-F DQ904582 Exiguobacteriuin sp. inialT T 621-G 1 DQ904583 Serratia sp. Serratia9T4l. Ru/inc/la 7. Yers 2 teriu,n 16T34, R,emerella ]4T 16 T 621-I DQ904584 Chrvseobacterium Chrvceohac sp. N.B 621-i DQ904585 Enterobacteriaceae Acinetobacter32T NJ 628-A DQ904586 Acinetobucter sp. 28. P. agglomerans T 24. N 628-B DQ904587 Pantoea E. cloacae2T agglomerans Citrobacterjreundii 2 N,T.B 628-C DQ904588 Pseudotnonas sp. Pseudomonas sp. toea T N 628-E DQ904589 Entembacter sp. Enterobacter4T, pan T 628-Fm Acinetobacter sp. terium 19T N 628-G DQ904590 Chryseobacterium Chryseobac sp. jam,laeT, peor,aeT 2. terrue N 628-H DQ904591 Paenibacillus polvmyxaóT52, poivmYxa 1 Paenibacillusl6T N 628-I DQ904592 Paenibacillus sp. 54 B 628-J" Enterobacter sp. Paenibacillusl6T44, Cohnella 3 B 628-K DQ904593 Paenibacillus sp. polymyxaOT48, jamilae T. iaeT T 628-L DQ904627 Paenibacillus terrae 1. peor polvmyxa 2, kribbensisT. dejeonensisT 1 = forward (5) sequence only; m =morphological ID: = reverse (3) sequence only. N=Nutrient agar; T=Tryptic soy agar; B=Brain heart infusion. + Isolated from cultures grown under anaerobic conditions with an Anerogen gas pack, in addition to being found in aerobic cultures. Superscript #T represents the number of type strains in same dade as the query sequence. Numbers associated with a species name correspond to the number of non-type strains in the same dade as query. For example. luteu S2T 42. t/iailandicus I means there were 2 type and 42 non-type strains with the luteus name in the same dade as the query sequence together with I thailandicus type strain.

483 Phytoparasitica 35:5. 2007

Table 1. Continued Sample GenBank Identity Type ( i ) and environmental isolates of Media Accession bacterial Species/Genera in dade with query used No. (BIB1) 719-A DQ904594 Pseudomonas sp. Pseudomonas8152 N 719-B DQ904595 Enterobacterjaceae N 719-C DQ904596 Pseudo,nonas sp. Pseudomonas381"52 N 719-1) "1 Pseudomonas sp. N 719-E DQ904597 Enterobacterjaceae N 719-F DQ904598 Pseudomonas sp. Pseudomonas6T52 N 719-G DQ904599 Pseudomonas sp. Pseudonionas48754 B 719-I-i DQ904600 Rahnella R. aquatilis2Tii B aqualilis 719-1 DQ90460I Pseudomonas sp. Pseudomonas48750 719-Jm B Chrvseobacte,-jum T sp. 719-K DQ904602 Pseudomonas sp. Pseudomonas47750 T 719-L DQ904603 Pseudomonas sp. Pseudomonas48T5O T 719-M DQ904628 Panroea agglomerans4T T,B agglomerans 719-Ntm Bacillus N megaterium 719-0 DQ904604 Paenjbacjllus sp. Paenjbacjllus I IT 44, Bacillus 3, cohnella 3 1 719-ptm Paenjbacjllus sp. I 719-Q DQ904605 Enterobacterjaceae T 719-R DQ904606 Rahnella R. aquatilis2T 8 T aquatilis+ 728-A DQ904607 Pseudomonas sp. Pseudomonas 48T 50 B 72813m Pseudomonas sp. B 628Jtm Enterobacter sp. B 728Crn Pseudomonas sp. TM 728Dm Pseudomonas sp. T,B 728-E DQ904608 Bacillus megaterium4T50 T megaterium 728Fm Pseudomonas sp. T 728-G DQ904609 Bacillus simplex simple.rT20, psychrosaccharolvticus 2, 1 butanoljvorans 728-Fltm Bacillus I megaterium 728-1 DQ9046 10 Bacillus megarerium3T43, flexus 7 N megaterium 728-J DQ90461 I Bacillus pumilus pumilus2T50 B 728-Kf DQ904612 Bacillus simplex simplex4T B 728-L DQ904613 Rahnella aquatilis2T7. Ewingella americana 3 N,T,B aquatilis m 1 = forward (5) sequence only; =morphological ID; r = reverse (3) sequence only. N=Nutrient agar; T=Tryptic soy agar: B=Brain heart infusion. + Isolated from cultures grown under anaerobic conditions with an Anerogen gas pack, in addition to being found in aerobic cultures. Superscript #T represents the number of type strains in same dade as the query sequence. Numbers associated with a species name correspond to the number of non-type strains in the same dade as query. For example. luteus2T 42. thailandicus I means there were 2 type and 42 non-type strains with the luteus name in the same dade as the query sequence together with 1 thailandicus type strain.

484 L.A. Lacey et al. Table 1. Continued Type (") and environmental isolates of Media Sample GenBank Identity used Accession bacterial Species/Genera in dade with query (BIBI) No. 728-M 1 DQ904614 Erwinia sp. Erwinia61 - 728Ntm Pseudo,nonas sp. - marcescens2T 50, ureilvticaT N,T,B 818-A DQ904615 Serratia marcesCeflS+ caicoaceticus5T N 818-B DQ9046 16 Acinetobacter calcoaceticus N 818Cm Enterobacter sp. megaterium2T 42.flexus 8 T 818-D DQ904617 Bacillus megaterium Enterobacter sp. T,13 818-Em T 81 8-ImChrvseobacterium sp. T 818-G m Cellulosimicrobium sp. Bacillus4IT NJ 818-H DQ904626 Bacillus sp. N DQ904618 Bacillus sp. Bacillus40T (thur,ng,ensis?) 818-1 N,T.B 818-J m Bacillus inegaterium NJ 8 18-Ktm Bacillus sp. Bacillus47T N 818-L DQ904625 Bacillus sp. T 818-Mm Bacillus pnegateriUm T 8 18-Ntm Bacillus megaterium Serratia3T T 818-P DQ904620 Serratia sp. T DQ904621 Pseudomonas sp. Pseudomonas46T 830-A B 830-Ctm Rahnella aquatilis+ N 830-Dr DQ904624 Enterobacteriaceae Enterobacteaceaen N 830-Etm Enterobacteriaceae 830-Ftm Bacillus - megaterium T,B DQ904622 Bacillus sp. 830-G T 830-Htm Bacillus megateriUm T 830lm Bacillus megateriUm N 830-Jtm Bacillus sp. o.rvtoca5T N,T.B 830-K DQ904623 Klebsiella oxvtoca N,T.B 830-Ltm Pseudomonas sp. 1 = forward (5) sequence only; m =morphological ID; = reverse (3) sequence only. N=Nutrient agar; T=Tryptic soy agar; B=Bram heart infusion. + Isolated from cultures grown under anaerobic conditions with an Anerogen gas pack, in addition to being found in aerobic cultures. Superscript #T represents the number of type strains in same dade as the query sequence. Numbers associated with a species name cot-respond to the number of non-type strains in the same dade as query. For example, luteus name in the same luteus2T 42, thailandicus I means there were 2 type and 42 non-type strains with the dade as the query sequence together with I t/iatlandicus type strain.

485 Phytoparasitica 35:5, 2007 TABLE 2. Bacterial families and genera represented in the 86 isolations [Numbers in parentheses represent the number of isolates for each genus. Seven isolates of Enterobacteriaceae could not be resolved below family.]

Bacjjlaceae Bad//is (22), Exiguobacterjum (2) Enterobacterjaceae Enterobacter (4). Erwinja (I), K/ebsjejla (I), Pantoea (2), Rahnel/a (4), Serratia (3) Flavobactenaceac Chrvseobacterjum (4) Microbacterjaceae Leucobacter (I) Mtcrococcaceae Mcrococcus (I) Moraxellaceae Acinetobacter (3) Pacnibacillaceae Paenjbacjllus (6) Promicromonosporaceac Ge//u/os imicrobjum (2) Pseudomonadaceae Pseudo,nonas (23)

DISCUSSION

Of the nine distinct species that were isolated in our survey, only two were found at both sites (B. megaterium, Pantoea agglomerans), two species were found only at the Hermiston site (Micrococcus luteus. Paenibacilluspolymyxa) and five species were seen only at Moxee (Bacillus pumilus, B. simplex, Klebsiella oxvtoca, R. aquatilis, S. marcescens). These differences may arise from non-overlapping sampling dates and habitat differences. The Hermiston site is a research farm where multiple insecticides have been tested over decades: it is located in one of the warmer growing areas in the Columbia Basin, and was sampled in May and June. The Moxee site is a research farm where insecticides have been used sparingly over the last 10 years, has a markedly cooler climate and was sampled in July and August. The soil type at the two locations is similar. The most commonly found bacterium in our survey. B. megaterium, has been utilized in numerous experiments where its plasmids have been manipulated to express a number of products (33). It is a spore-forming, aerobic bacterium found in a variety of settings including soil, seawater, sediments, rice paddies, dried food, honey and milk (33). B. megaterium is biochemically versatile and used in many industrial applications and as a cloning host for expression of intact foreign proteins (33). It has the additional advantage of growing in basic nutrient media without any added growth factors. Most B. megaterium strains usually contain four or more plasmids (29). Some of these have been modified to express toxins from the insecticidal bacteria B. thuringiensis (1,4,30) and B. sphaericus (11) and toxins from Clostridium difficile (6). As a naturally occurring bacterium in the potato agroecosystem with the potential to express bacterial toxins. B. nIegaterium appears to be an ideal candidate for production of gene products with control potential for L. canus. The second most frequently isolated bacterium in our survey, R. aquatilis, is a widely distributed Gram-negative, N2-fixing, facultatively anaerobic species that has been isolated from fresh water, soil and the rhizosphere of several plants (5,12). Because of its antagonistic activity, it has been used as a microbial control agent of certain plant pathogens (10,23). The natural occurrence of R. aquatilis in potato agroecosystems and its rhizosphere colonizing ability could provide unique options for wireworm control. For example. R. aquatilis that expressed wireworm-active toxin could be used as a potato seed treatment, as it is likely to remain in the rhizosphere of the growing plants. Alternatively or additionally, it could be used in conjunction with trap crops that are attractive to wireworm larvae.

486 L.A. Lacey et al. This bacterium is common in the wheat rhizosphere (12) and germinating wheat is highly attractive to wireworm larvae (14). Planted rotationally with potato or other root crops that are attacked by wireworm, wheat treated with modified R. aquatilis could help to eliminate tuber-damaging larvae before potatoes are planted. Other bacteria found in our survey, such as S. marcescens and other Serratia species. are also reported to contain plasmids with potential for expressing toxins and other products. Serratia species are commonly found in the guts of insects (17,24.32), but are not normally pathogenic unless they are introduced into the hemocoel. An exception is S. entoinophila, which colonizes the gut and starves the larvae of grass grubs, Costelvtra zealandica (White) (16). Hurst etal. (15) reported cloning antifeeding genes of S. entonophi1a into Escherichia coli, and of other Serratia species that exerted strong antifeeding activity and often led to rapid death of infected grass grub larvae. The potential of these genes expressed in a bacterium that is fed upon by wireworms or is resident in the gut of L. canus warrants further attention. Although S. entoinophila is specifically active in C. zealandica, it may also be worth investigating the microbial control activity of other Serratia species that are engineered with antifeeding genes of S. entoinophila for their ability to control L. cantis and other insects that feed on potato tubers, such as flea larvae (Epitrix spp.) and potato tuber moth (Phthorimaea operculella (Zeller). While feeding on host plants, wireworms ingest soil from the surface of tubers and other roots. Bacteria are the most numerous of the microorganisms found in soil (27). Consequently, most of the bacteria isolated from the guts of L. canus are probably ingested transients. Bacteria that reproduce in the out will be the best candidates for paratransgenesis. However, obligate gut residents - in contrast to the more facultative or transient residents - are likely to be fastidious and missed by our media-first isolation methods. While a cloning-first method of gut flora identification is more likely to uncover the more fastidious species (36), their manipulation, propagation, and subsequent use as infective agents will require complex media and handling and a significant increase in our knowledge of their biological requirements. Ideally, ongoing studies will allow us to identify species that reproduce in the gut of L. can us, yet are not overly fastidious. Once candidate bacteria that reproduce in the gut or colonize the potato rhizosphere are identified, further research will include discovery of genes that express toxins or other factors detrimental to wireworms, identification of plasmids in the bacteria associated with L. canus and determining which can be modified for control of this beetle. Ultimately, the inoculation of bacteria expressing beetle-active toxins into the potato agroecosystem could be done by applying them to the surface of potato seed pieces or other attractive substrates such as the bait balls used in this survey. A naturally occurring bacterium found in the gut and/or environment of a targeted insect that is modified to express toxins or other detrimental substances could provide certain advantages over inundatively applied microbial control agents, particularly within soil habitats. Persistence and recycling of the bacterium and secretion of detrimental substances within the gut of targeted insects would be especially advantageous. Plasmids expressing toxin-producing genes may be better suited to wireworm biology. Bacillus thuringiensis toxins with activity against other coleopterans in the families Chrysomelidae and Scarabaeidae have been reported (3. 19,22). The Cry3Aal I toxin identified by Kurt et al. (19) is of particular interest as a candidate for wireworm control due to its fairly broad coleopteran host range and expression in another bacterium. Bacillus subtilis.

Phvtoparasitica 35:5, 2007 487 qRs

ACKNOWLEDGMENTS

We are grateful to David Horton and Kathie Johnson, USDA-ARS Wapato. for providing bait balls and wireworni sampling advice. Our thanks are expressed to Drs. Horton, Steve Garczynski, Joel Siegel, Peter Landolt and Don Hostetter for reviews of our manuscript and for providing useful comments; and to Dr. George Clough and Mr. Jerry Gefre for providing information and logistic support at the Hermiston and Moxee stations, respectively.

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