Experimental Pathogenicity of Erwinia Aphidicola to Pea Aphid, a Cyrthosiphon Pisum
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J. Gen. App!. Microbiol., 43, 363-367 (1997) Experimental pathogenicity of Erwinia aphidicola to pea aphid, A cyrthosiphon pisum Hosami Harada*,t and Hajime Ishikawa Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Bunkyo-ku, Tokyo 113, Japan (Received October 17, 1997; Accepted December 11,1997) Pea aphids were reared aseptically in the laboratory and fed on strains of Erwinia aphidicola, Er- winia herbicola, Klebsiella pneumoniae, Escherichia coil and bacterium W, an aphid enterobac- terium, by mixing with a synthetic diet to determine whether these bacteria have pathogenicity to the insect. It turned out that Er. aphidicola and Er. herbicola grew in aphid gut, while K. pneumo- niae, Es. coil and bacterium W were effectively eliminated. In the case of Er. aphidicola, ingestion of a single dose of 103 CFU/ml was enough to infect aphid gut and to prevent post-final ecdysis growth of the insect. As a result, the body weight of the infected aphids was significantly reduced (p<0.01). A concentration of 105 CFU/ml of Er. aphidicola caused aphid mortality. In contrast, Er. herbicola demanded 105 CFU/ml to colonize in aphid gut, and seemed to have no effect on insect health. Key Words biological control; Erwinia aphidicola; Erwinia herb/cola; pathogenicity Aphids are one of the most important pest insect the family Enterobacteriaceae (Unterman et al., 1989). groups in agriculture in temperate climatic zones. In addition to this highly-conserved type of endosym- These insects debilitate their host plants both directly biont, many lineages of aphids harbor other types of as sap feeders, toxifiers and pollutive excreters, and intracellular symbionts, so called secondary sym- indirectly as vectors of plant virus diseases. In order to bionts, which are distinct members of the family Enter- prevent or diminish these negative effects of aphids, obacteriaceae (Buchner, 1965; Fukatsu and Ishikawa, many attempts have been made to control aphids by 1993; Fukatsu, T., pers. commun.). It has been sug- chemical, biological or integrated control methods. gested that occurrence of the secondary symbionts Most attempts at the biological control of aphids in- does not have a clear correlation to the phylogeny of volved the introduction of insect predators such as the host insects (Fukatsu, 1994), implying that the coccinellids (Carver, 1989). Aphid pathogenic fungi members of the family Enterobacteriaceae infect were also studied as a candidate of agents for the bio- aphids repeatedly. logical control of aphids (Latge and Papierok, 1989). In a previous study, we demonstrated, that the pea So far, bacterial infections to aphids have not been aphid Acyrthosiphon pisum harbors at least three systematically demonstrated, whereas aphids are groups of bacteria belonging to the family Enterobac- known for their general affinity for bacteria related to teriaceae in its gut, and tentatively named them bac- the family Enterobacteriaceae. In the meantime, al- terium T, W and X (Harada et al., 1996). One group, most all the aphid species harbor mutualistic en- bacterium T, was definitively identified as Erwinia her- dosymbionts in mycetocytes, which are specifically b/cola, which occurs usually on the leaf of healthy differentiated cells to accommodate them (Buchner, plants. For bacterium X, the predominant species in 1965). The symbionts are members of the y-3 subdivi- aphid gut, the establishment of a new species, Erwinia sion of the class Proteobacteria and closely related to aphid/cola, was proposed based on the biochemical characteristics and results of DNA-DNA hybridization tests (Harada et al., 1997). * Address reprint requests to: Dr. Hosami Harada, Department of In this study, we investigated the effects of ingestion Biological Sciences, Graduate School of Science, The University of of members of the family Enterobacteriaceae on pea Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113, Japan. aphids which had been reared aseptically in the labo- t Present Address: Bio-oriented Technology Research Advance- ratory. It was revealed that Er. aphidicola has patho- ment Institution, Nisshin, Ohmiya 331, Japan 364 HARAOA and ISHIKAWA Vol. 43 genicity to aseptic aphids. teria were enumerated by dilution plating on LB agar after 24-h incubation to confirm the number in inocu- Materials and Methods lum. The synthetic diet used here was that of Febvay et al. (1988) with modifications as described by Sasaki Insects. Aphids used in this study were obtained et al. (1991). Bacteria were suspended in 400 µl of from a long-established parthenogenetic clone of pea synthetic diet dyed with neutral red, and enclosed in aphids, Acyrthosiphon pisum (Harris), which were stretched Parafilm membranes as previously de- reared on young broad bean plants, Vicia faba (L.), at scribed (Srivastava and Auclair, 1971). Aphids born 20°C under a long-day regime of 18h light and 6 h and reared on the plant for 4-6 days, which were dark. A colony of the aphids without gut microbes was third-instar nymphs, were transferred to the synthetic established by keeping the insects on plants grown diet and fed on it for 24 h to ingest bacteria. Aphids, from surface-sterilized seeds on agar media in steril- which turned red by ingesting the synthetic diet, were ized test tubes for more than 100 generations (Harada transferred to the plant and reared on it for 3 days to and Ishikawa,1993). adulthood. All the experiments were carried out at To minimize alate production in the aphid colony, a 20°C with a photoperiod of 18 h. small number of adults (5-10 individuals) were al- Bioassays. Aphid mortality was scored 1 day after lowed to deposit nymphs for 48 h on a plant. The par- final ecdysis. Survived aphids were measured for their ents were removed and the nymphs found at this time body weight and checked for infection with bacteria were considered to be 0-2 days old. as follows. The insects were surface-sterilized by dip- Bacterial strains. All cultures used in this study ping in 70% ethanol for 5 min, washed in Calson's so- were maintained in LB nutrient media containing 30% lution [0.7% NaCI (w/v), 0.02% KCI (wlv), 0.02% glycerol at -80°C. Cultures were grown at 30°C in LB CaCl2.2H2O (w/v), 0.01% MgCl2.6H2O(w/v), 0.02% nutrient liquid media or on LB agar. Erwinia aphidicola NaH2PO4 (wlv), 0.012% NaHCO3 (w/v), 0.8% glucose (IAM 14479T, 14480, 14481, 14482 and 14483), Er- (wlv)] and then lightly homogenized in 0.2 ml of the winia herbicola (191) and bacterium W (lAM 14478T) same solution. An aliquot of the homogenate was were isolated from aphid gut, and identified by bio- spread on LB agar and incubated overnight at 28°C to chemical tests and DNA-DNA hybridization (Harada et detect bacteria. al., 1996; Harada et al., 1997). Erwinia herbicola (JCM 7000) was obtained from the Japan Collection of Mi- Results croorganisms. Escherichia coli XL1-blue is a deriva- tive of the strain K12. Klebsiella pneumoniae (6081) is The effects of Er. aphidicola and Es. coli a nonpathogenic strain (Dohra et al., 1997). Log phase cultures of Er. aphidicola and Es. coli Inoculation of bacteria to aphids. A log phase cul- were collected and diluted in the synthetic diet to 103, ture of the bacteria, whose growth was estimated in 105, 10' and 109 CFU/ml, and third-instar nymphal terms of light absorbance, was centrifuged at aphids were fed on them. Aphids which were fed on 3,000 rpm for 10 min, and the precipitate was sus- Er. aphidicola, began to die soon after final ecdysis. At pended and diluted in a synthetic diet for aphids. Bac- concentrations higher than 105CFU/ml, almost all the Table 1. Effects of Erwinia aphid/cola and Escherichia coli on aphids. 1997 Pathogenicity of E. aphid/cola to aphid 365 Fig. 1. Bacteria detected from aphids which fed on the synthetic diet inoculated with Erwin/a aphid/cola (A) and Es- cherichia coli (B). Numerals represent the number of bacterial cells inoculated in the synthetic diet (CFU/ml). Table 2. Mortality of aphids caused by Erwinia aphid/cola strains. Table 3. Infection of bacteria to aphids. aphids were dead a day after final ecdysis. Though about a half of the aphids survived at 103 CFU/ml, growth after final ecdysis was retarded and their body Infection of other bacteria to aphids weight was significantly reduced (p<0.01). In contrast, Infection by other bacteria examined in this study is no effect was observed when aphids were fed on Es. summarized in Table 3. Each species was diluted in coli (Table 1). In order to determine whether the the synthetic diet to a final concentration of 105 CFU/ml aphids were infected with these bacteria, the insects and subjected to the infection experiment. About 20- that survived were lightly homogenized in Calson's so- 25 aphids were tested for each bacterial species. lution after surface-sterilization and one-5,000th of the Since no mortality or growth retardation was ob- homogenate was spread on LB agar. A large number served, we selected six aphids randomly from each of bacterial colonies were detected from the aphids group and examined whether the aphids were infected which were fed on Er. aphid/cola at any concentration or not. It turned out that Er. herb/cola, as well as Er. examined (Fig. 1 A). In contrast, no colonies were de- aphid/cola, has infectious activity to the aphid gut. tected from the aphids that had ingested Es. coli at More than a thousand bacterial colonies were de- 103 and 105 CFU/ml. Only a small number of colonies tected from the two aphid groups which were fed on were observed when the aphids ingested Es.