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28 PHOTORHABDUS AND XENORHABDUS: A NEW SOURCE OF USEFUL COMPOUNDS FOR BIOLOGICAL CONTROL A. Ragni and F. Valentini BioTecnologie BT srl Pantalla di Todi 06050 Perugia Italy Tel: ++39 – 075 - 895091 Fax: ++39 – 075 – 888 776 E-mail: aragni.bt@parco3a.org IntroductionThe genera Photorhabdus and Xenorhabdus are symbiotic Enterobacteriaceae bacteria associated with entomopathogenic nematodes of the genera Heterorhabditis and Steinernema, respectively. The characteristics of both Photorhabdus and Xenorhabdus genera have been reviewed by Forst & Nealson (1996), and by Forst et al. (1997). Recently Bowen et al. (1998a; 1998b) and Blackburn et al. (1998) discovered and characterized an insecticidal toxin complex that it is secreted into the culture medium by Photorhabdus luminescens strain W-14. Except for this specific strain, the insecticidal capability of entomopathogenic soil nematode symbiotic bacteria has been reported only when carried into the insect by the nematodes or artificially injected into their haemocel. This work reports the isolation of a unique strain Photorhabdus sp., named XP01, selected among 12 Photorhabdus and Xenorhabdus isolates, that displays an insecticidal activity when fed to the neonate larvae of several Lepidoptera pests.Materials and Methods. Bacteria isolation The bacteria strains tested were isolated as described by Akhurst (1980), from cadavers of Galleria mellonella infected with Heterorhabditis spp. or with Steinernema spp. strains belonging to the collection of entomopathogenic nematodes held at BioTecnologie S.r.l (BT) laboratories. A list of the isolated strains is shown on Table 1. Cultures were maintained by weekly subculturing on Nutrient Agar plates and incubated in the dark at 25°C. Inocula for liquid cultures were prepared as fermented broth aliquots containing glycerol at a final concentration of 15% (v/v) and stored at -80°C. Bacterial preparations Whole cultures were obtained by growing the strains in a Casein peptone plus yeast extract liquid medium in the dark at 25°C and shaking at 180 rpm , for a period of either 19 or 24 hours. Technical powder of XP01 was prepared by freeze-drying the pellet obtained by centrifugation the bacterial culture at 6,238 x g for 40 min. The pellet was washed three times in a neutral Ringer’s solution prior freezing. Freeze-drying resulted in cells dead since plating the resuspended technical powder gave no colony growing on Nutrient Agar. Insects rearing and bioassay techniques Colonies of Mamestra brassicae, Cydia molesta, Spodoptera littoralis, Helicoverpa armigera, Scotia segetum and Agrotis ipsilon are continuously reared at BT insectarium under controlled environmental conditions and fed with semi-synthetic diets. Insecticidal activity of bacterial suspensions was measured by using an overlay technique bioassay. Bacterial preparations at different serial dilutions were applied in 50 µl aliquots in the wells of a diet bioassay trays (surface area 175 mm 2 ) containing 1 g of an insect meridic diet. As control, the insect diet was treated with deionized sterile water. After drying, one unfed 1st instar larva (neonate) was placed in each well. Each treatment was applied to 32 insects. Mortality was scored after 5 days of incubation at 27°C. Results. Preliminary Screenings A total of 12 Photorhabdus and Xenorhabdus bacterial isolates were assayed for insecticidal activity against neonates of Mamestra brassicae and of Cydia molesta in a surface contamination assay. As shown in Figure 2, neonates of Cydia molesta were more susceptible than those of Mamestra brassicae when exposed to fermented broths of Photorhabdus and Xenorhabdus isolates. Treatments with XP01 and XP04 fermented broths resulted in 100 % mortality of C. molesta neonates. In this species the mortality rate ranged from 25 to 41% for treatments with XP136, XP1007 and XP127. Low activity (from 0% to 19% mortality) against C. molesta neonates was recorded when treatments were done with the remaining bacterial strains. A 100% mortality of Mamestra brassicae neonates was observed when the insects were treated with XP01. A lower mortality equal to 74% was observed when XP04 was used. A residual activity (5% larval mortality) was recorded when treatment was done with strain XP10. PDF creato con FinePrint pdfFactory versione dimostrativa http://www.secom.re.it/fineprint 132
After this preliminary screening and due to the high activity of XP01 against both larvae insect tested, this strain was selected for further characterization of its insecticidal activity. The XP01 activity was further tested on several other lepidopteran species. For convenience purposes only macrolepidopteran species were used in these assays. Results of this second screening are shown in Figure 3. The whole culture of XP01 showed the capability to kill all the five species treated, being most effective against M. brassicae, S. segetum and A. ipsilon. The insecticidal activity of freezed dried technical powder, made from the cell pellet of fermented broth, was tested against neonates of M. brassicae. As seen before with other bacterial preparations, the observed mortality of insects was proportional to the applied dose (Figure 4). Discussion. Photorhabdus and Xenorhabdus species are bacteria known to live in symbiotic association with entomopathogenic soil nematodes. Mechanisms involved in the pathogenicity of these bacteria resulting from their release by entomopathogenic nematodes in insects, have been extensively studied. A great deal of variability in the bacterial dose needed to kill different insects has been reported. The effect both depending on the bacterial strain used as well as the target insect studied. The present study describes the discovery of a newly characterize strain of Photorhabdus luminescens, denominated XP01, which is highly toxic when directly administered by food contamination assays to neonates larvae of several Lepidopteran pests including Mamestra brassicae and Cydia molesta. It was demonstrated that the toxic effect of XP01 is maintained even when a technical powder is used. Since technical powders do not contain alive cells, this result indicates that once the toxin has been synthesized alive bacterial cells are not further required for activity. This characteristic of XP01 insecticidal capability is similar to what has been described for Bacillus thuringiensis toxins (Schnepf et al., 1998). The discovery of the insecticidal activity of Photorhabdus luminescens strain XP01 indicates the existence of a presumably new family of entomotoxic proteins present in Photorhabdus and probably Xenorhabdus spp. These insecticidal toxins could be used as an active bio-pesticide ingredient in a similar manner to the delta-endotoxins of B. thuringiensis. This new source of natural toxins could help both the fight against the development of insects resistance as well as serve as sources of novel specific activities in combination with other known biopesticides.Acknowledgements We thank G. Flek for rearing insects for tests, L. Quattrocchi and R. Lorenzini for their assistance in microbiology and bioassay works. References Akhurst, R.J. 1980. Morphological and Functional Dimorphism in Xenorhabdus spp., of bacteria symbiotically associated with insect pathogenic nematodes Neoplectana and Heterorhabditis. J. Gen. Microbiol. 121: 303- 309. Blackburn, M., E. Golubeva, D. Bowen, and. R.H. Ffrench-Constant. 1998. A novel insecticidal toxin from Photorhabdus luminescens, toxin complex a (Tca), and its histopathological effect on the midgut of Manduca sexta. Appl. Environ. Microbiol. 64: 3036-3041. Bowen, D.J., T.A. Rocheleau, M. Blackburn, O. Andreev, E. Golubeva, R. Bhartia, and R.H. Ffrench-Constant. 1998a. Insecticidal toxin from the bacterium Photorhabdus luminescens. Science. 280: 2129-2132. Bowen, D.J., and J.C. Ensign. 1998b. Purification and characterization of a high-molecular-weight insecticidal protein complex produced by the entomopathogenic bacterium Photorhabdus luminescens. Appl. Environ. Microbiol. 64: 3029-3035. Forst, S. and K. Nealson. 1996. Molecular biology of the symbiotic-pathogenic bacteria Xenorhabdus and Photorhabdus spp. Microbiol. Rev. 60: 21-43. Forst, S., B. Dowds, N.E. Boemare, and E. Stackebrandt. 1997. Xenorhabdus and Photorhabdus spp.: bugs that kill bugs. Annu. Rev. Microbiol. 51: 47-72. Schnepf, E., N. Crickmore, J. Van Rie, D. Lerecluse, J. Baum, J. Feitelson, D.R. Zeigler, and D.H. Dean. 1998. Bacillus thuringiensis and its pesticidal crystal proteins. Microbiol. Mol. Biol. Rev. 62: 775 -806. PDF creato con FinePrint pdfFactory versione dimostrativa http://www.secom.re.it/fineprint 133
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International Congress BIOLOGICAL P
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15:40-16:20 G. Lozzia Università d
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MAIN SPEAKERS • V. Rotondo FIAO,
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RELIABILITY AND PROSPECTIVES FOR TH
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LOZZIA G.C., RIGAMONTI I.E., 1994 -
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DIFESA DELLE COLTURE DAI PATOGENI F
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THE FUNGAL PHYTOTOXINS AND THEIR PO
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NEED FOR RESEARCH AND APPROPRIATE A
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PRESENCE OF OCHRATOXIN IN EXPERIMEN
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BIOPESTICIDES: EVALUATION PROCESS F
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californicus (fitoseide predatore)
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Figure 3. Effect of Pseudomonas sp.
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(mg kg-1) (mg kg-1) 140 120 100 80
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Recovery and repeatabilty tests wer
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