INTERNATIONALJOURNAL OF SYSTEMATICBACTERIOLOGY, Jan. 1988, p. 1-6 Vol. 38, No. 1 OO20-7713/88/01OO01-06$02.W/O Copyright 0 1988, International Union of Microbiological Societies Serratia entomophila sp. nov. Associated with Amber Disease in the New Zealand Grass Grub Costelytra zealandica PATRICK A. D. GRIMONT,l* TREVOR A. JACKSON,' ELISABETH AGERON,l AND MICHAEL J. NOONAN3 Unit&des Enttrobacte'ries, Institut National de la Sante' et de la Recherche Me'dicale Unitt 199, Institut Pasteur, F-75724 Paris Ce'dex 15, France'; Agricultural Research Division, Ministry of Agriculture and Fisheries, Lincoln, Canterbury, New Zealand2; and Department of Agricultural Microbiology, Lincoln College, Canterbury, New Zealand3 Serratiu entomophila sp. nov. is a homogeneous deoxyribonucleic acid relatedness group most closely related to Serratia ficaria and Serratia marcescens. All 19 strains studied resembled S. marcescens (the phenotypically closest species) by their inability to ferment or utilize L-arabinose, L-rhamnose, D-melibiose, dulcitol, and D-raffinose but differed from S. marcescens by their inability to ferment or utilize sorbitol and their lack of lysine and ornithine decarboxylases. All strains utilized itaconate, a unique characteristic among Serratia species. Two biotypes could be distinguished. Strains of biotype 1 utilized D-arabitol but not L-arabitol or D-xylose. Strains of biotype 2 utilized L-arabitol and D-xylose but not D-arabitol. S. enturnuphila has been isolated from insect larvae and water. Most strains were isolated in New Zealand from the grass grub Costelytra zealandica infected with amber disease. None of the strains were isolated from infected humans, animals (other than insects), or plants. The type strain is strain AIT (ATCC 43705T). The grass grub Costelytra zealandica White (Coleoptera; (A2a), 286 (A2b), 253 (A3b), 326 (A4a), 4466 (A4b), 246 (A5), Scarabaeidae) is a major pasture pest in New Zealand and 3780 (A6), 268 (A8b), 81, and 3583 (TCT). The type or causes damage estimated at up to $100 million (N.Z. cur- reference strains of other species used for comparison are rency) per year. Larvae of Costelytra zealandica feed on the same as used in earlier works (1, 9, 10). grass, clover, and other plant roots. Typically, their popu- DNA relatedness study. Published procedures to extract, lations grow to a peak in 4 to 6 years after the pasture is sown purify, and shear unlabeled DNAs were used (2). The and then collapse. Grass grub population decline was found procedures used for in vitro labeling of DNA with tritium- to be associated with the .presence of a disease called amber labeled nucleotides and for hybridization experiments (nu- disease (previously referred to as honey disease [22]). The clease S1-trichloroacetic acid method) have been described major bacterial agent of honey disease, a Serratia sp. (pre- previously (12). The denaturation temperature ( Tm), at viously referred to as Hafnia alvei [22] or Serratia marino- which 50% of the reassociated DNA became hydrolyzable rubra [21]), was isolated from naturally infected larvae (22) by S1 nuclease, was determined by the method of Crosa et and shown experimentally to produce the disease when al. (3); AT, is the difference between the T, of the heterol- transmitted to healthy larvae (22). Orally infected larvae stop ogous DNA pair and the T, of the homologous DNA. feeding within a few days, clear the gut, develop an amber G+C content determination. The denaturation tempera- discoloration, and gradually lose weight until death occurs 4 tures of 50-pg/ml DNA solutions in 0.1~SSC buffer (Ix to 6 weeks later (22) (Fig. 1). The bacteria colonize the gut SSC is 0.15 M NaCl plus 0.015 M trisodium citrate) were and cause disease symptoms without invading the hemo- measured with a Gilford spectrophotometer (Gilford Instru- coele (a body cavity largely filled with blood). Another ment Laboratories, Oberlin, Ohio). The guanine-plus-cy- bacterium, Serratia proteamaculans (previously referred to tosine (G+C) contents of DNAs were determined from the as Serratia liquefaciens [21]) was also shown to cause amber denaturation temperatures (17). The DNA of Escherichia disease in the grass grub. coli K-12 was used as a standard (G+C content, 51.2 mol%). The purpose of the present work was to determine, by Assimilation tests. Carbon source utilization tests were deoxyribonucleic acid (DNA) relatedness and extensive done by using specially manufactured API strips (API Sys- phenotypic characterization, the taxonomic position of the tem, La Balme les Grottes, France) which contained pure Serratia sp. causing amber disease in the grass grub. The carbon sources and were similar to commercial API CH, outcome of the study is the description of a new species for API AO, and API AA galleries (3,except that the total which the name Serratia entomophila is proposed. number of tests was 114 carbon sources (listed in the species description below). The minimal medium was supplied by MATERIALS AND METHODS API System. The strips were inoculated as described earlier (l),incubated at 3WC, and examined daily for growth for 4 Bacterial strains. The sources of S. entomophila strains days. are given in Table 1. Grass grub strains were isolated from Conventional biochemical tests. All tests were incubated at the midgut of larvae after dissection under sterile conditions 30°C unless otherwise stated. These tests included produc- (21). The following 13 Serratia $curia strains were repre- tion of gas and H,S in glucose-lactose-iron agar (Diagnostics sentative of the four known serotypes of that species (7): Pasteur, Marne-la-Coquette, France), urea hydrolysis, and 2602, 4024T (T, type strain), 4026, 4028, 4034, 4037, 4038, indole production from tryptophan in urea-indole medium 4039, 4600, 4738, 4739, 4747, and 4750. The following 12 (Diagnostics Pasteur), gelatin hydrolysis (film method) (16), Serratia marcescens strains represented 11 of the known the P-glucuronidase test (14), the y-glutamyltransferase test biotypes (indicated in parentheses): 5 (Ala), 328 (Alb), 504T (6), growth on caprylate-thallous agar (20), growth in tryptic soy broth (Diagnostics Pasteur) at different temperatures, * Corresponding author. growth in peptone water (Bacto-Peptone [Difco Laborato- 1 2, GRIMONT ET AL. INT. J. SYST.BACTERIOL. bles 2 and 3) since some DNA preparations yielded either high relative binding ratios (up to 72%) or relatively low ATm values (e.g., 5°C). However, high relative binding ratios matched with high AT, values, and low AT, values matched with low relative binding ratios. The average relatedness of S. Jicaria strains to labeled DNA from S. entomophila AIT was 47% at 60°C and 58% at 70°C. The corresponding average ATm values were 6.7"C (hybridization at 60°C) and 7.5"C (hybridization at 70°C). The average relatedness of S. entomophila strains to labeled DNA from S. Jicaria 4024T was 56% at 60°C and 62% at 70°C. The corresponding average AT,,, values were 7°C (hybridization at 60°C) and 6.6"C (hybridization at 70°C). The other Serratia species were 28 to 48% and 14 to 38% related to S. entomophila at 60 and 70"C, respectively. FIG. 1. Second-instar larvae of C. zealandica. Healthy larva The following other members of the Enterobacteriaceae with dark gut on left, translucent larva showing symptoms of amber were 1 to 25% related to S. entomophila A1 at 60°C: disease on right. Budvicia aquatica 20186T, Cedecea davisae 5T, Citrobacter freundii 460-61, Edwardsiella tarda 10396=, Enterobacter aerogenes AIT, Enterobacter agglomerans E20T, Entero- ries, Detroit, Mich.], 10 g; distilled water, 1 liter; pH 7) bacter agglomerans I11 1429-71, Enterobacter agglomerans containing 0, 2, 6, or 8% (wthol) NaC1, and the Voges- VII 6003-71, Enterobacter amnigenus 78-79, Enterobacter Proskauer test, using the modification of Richard (19). All cloacae 1347-71, Enterobacter gergoviae 2-78, Enterobacter other tests were done by using procedures described else- intermedium 33422, Enterobacter sakazaki 4562-70, Erwinia where (9). amylovora EA178, Erwinia carnegieana EC186, Erwinia Production of enterobacterial common antigen. Production carotovora 495, Erwinia chrysanthemi SR32, Erwinia cypri- of enterobacterial common antigen by strain AIT was stud- pedii EC155, Erwinia mallotivora 2851T, Erwinia nigrifuens ied by using a previously described hemagglutination test EN104, Erwinia rhapontici ER106, Erwinia salicis ES102, (15, 18). Escherichia coli K-12, Ewingella americana C24, Hafnia Pathogenicity tests. For each strain tested, 30 grass grub alvei 329-73, Klebsiella pneumoniae 2, Proteus mirabilis larvae were incubated at 20°C for 14 days in vials of 20 g of PR14, Providencia stuartii 2896-6gT, Rhanella aquatilis 77- soil (containing about 1.5 x 10" bacteria) per larva. The lljT, Salmonella typhimurium LT2, Shigella boydii 1610-55, detailed method of the test has been published previously Xenorhabdus luminescens Hb, and Yersinia ruckeri 4535-69. (21). Among these non-Serratia species, the highest percent re- latedness was with E. carotovora (25%), and the lowest (1%) RESULTS was with P. stuartii. DNA-DNA hybridization. Several samples of DNA from S. DNA base composition. DNAs from strains AIT and A14 entomophila AIT and S.Jicaria 4024T were labeled, and the contained 57.6 and 58.0 mol% G+C, respectively (averages S 1 nuclease-resistant core (in the incubated control tubes of three determinations). containing only denatured labeled DNA) was 6 to 10% at Phenotypic characterization. Characteristics that were either 60 or 70°C. The Tm of homoduplexes (in 0.42 M NaC1) common to all strains studied are given below in the species was 95.2 to 99°C (strain Al) or 96.0 to 97.7"C (strain 4024). description. Characteristics that varied among the 19 strains All strains of S. entomophila studied were more than 74% studied are given in Table 4. Although all strains grew on related to strain AIT at both 60 and 70°C (Table 2) with caprylate-thallous agar (a medium containing yeast extract) insignificant divergence (AT, close to OOC).