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27 ECO-TOXICITY OF THE ENTOMOPATHOGENIC BACTERIUM-NEMATODE SYMBIOTIC COMPLEX TOWARD NON-TARGET ORGANISMS M. Ricci, M. Colli, R. Barcarotti and A. Ragni BioTecnologie BT srl Pantalla di Todi 06050 Perugia Italy Tel: ++39 – 075 - 895091 Fax: ++39 – 075 – 888 776 E-mail: mricci.bt@parco3a.org Introduction In agriculture, the use of bio-control agents is growing; this is due, in particular, to their ability to avoid pest’s resistance and to their friendly environmental impact. A mini-review of the entomopathogenic bacterium-nematode symbiotic complex toward non-target organisms is presented; bibliographic data and original data coming from research are shown. In particular it is mentioned a work which first demonstrated specificity of the entomopathogenic bacterium-nematode symbiotic complex toward target organisms (insects). The present work concerns three sections related to the eco-toxicity of entomopatogenic nematodes: a) Short review; b) Demonstration of entomopathogenic nematodes host specificity; c) Laboratory results toward nontarget hosts: Earthwoms, Scorpions and Millipeds. a) Short review Poinar (1990) reported that although Steinernematids and Heterorhabditids are considered entomopathogenic nematodes because all natural infections have involved insects, they do have the ability to enter and kill noninsect invertebrates, at least in the laboratory (Wayne et al., 1987). Steps of their infection process are normally completed in most insects but various obstacles to their completion occur with many non-insect invertebrates (Wayne et al., 1987). The effect of these nematodes on vertebrates is of a great concern. Off all the vertebrates (among the following classes: Pisces, Reptilia, Amphibia, Aves and Mammalia) thus far challenged with infective stage of Steinernematids and Heterorhabditids, only young tadpoles of frogs and toads were vulnerable. But the reason was given to the fact that when penetrating into the host the nematodes were carrying foreign bacteria. b) Demonstration of entomopathogenic nematodes host specificity ( Ricci et al., 1994) Introduction Entomopathogenic nematode infective juveniles (IJs) locate and parasitise potential hosts. The success of this process depends with suitability of the host. Ricci et al. (1994) and Lewis et al. (1996), demonstrated that a nematode's subsequent degree of attraction to CO2, stimulated by contact with the cuticle of an arthropod, should signify that the exposed IJs recognize it as a potential host. The level of stimulation caused to S. carpocapsae, a nematode that forages primarily by ambushing passing hosts, by 12 potential hosts (3 Lepidoptera, 4 Coleoptera, 1 Orthoptera, 1 Blattodea, 1 Diptera and 2 non-insect arthropods) was compared. To determine whether arthropods that stimulate a high level of attraction of IJs are suitable hosts, three parameters were tested: nematode induced mortality, nematode invasion rate and reproductive potential. Materials and methods Behavioral Recognition Assay: nematodes were exposed for a certain time to the cuticle of different insects or non-insects or inert materials, then exposed to chemical volatile cues emitted by the insects Galleria mellonella in an apparatus called Agar Plate Arena (see Figure 1). After a certain time the nematodes that accumulated in the proximity of the cue emissions, were collected and counted. Nematode Infectivity Assay: host mortality was recorded after 2 days in Petri dishes at the concentrations of 200 and 500 nematodes per host. Nematode Invasion Assay: Nematodes that penetrated the host during the Infectivity assay were counted. Reproductive Potential Assay: pre-weighed hosts were exposed to 500 IJs. After 2 days the cadavers were transferred on a nematode collecting trap. Every 2 days, the emerged IJs were collected until the exploitation of all the host tissues was completed. PDF creato con FinePrint pdfFactory versione dimostrativa http://www.secom.re.it/fineprint 128
Results and Discussion S. carpocapsae was differentially stimulated to be attracted to G. mellonella volatiles by the contact of the cuticle of different arthropods. Agrotis ipsilon pupa, Leptinotarsa decemlineata, Isopoda sp., Blattella germanica, Musca domestica and Diplopoda sp. were not significantly different from the controls. A. ipsilon larva, Tenebrio molitor, Acheta domesticus, G. mellonella, Diabrotica virgifera virgifera and Popilia japonica induced a significantly higher "activation" to S. carpocapsae IJs compare to the controls. It seems that S. carpocapsae can discriminate even among different stage of the same species in fact the value of A. ipsilon larva is more than twice that of the pupa. The same hosts that induced the higher attraction to the volatiles were more susceptible to the nematodes. The nematodes, established (penetration rate and reproduction index) better in the group of arthropods that where better stimulated by G. mellonella volatiles. It has been demonstrated that, at least one entomopathogenic nematode, S. carpocapsae, could discriminate among suitable and not suitable hosts. Figure 1: Agar Plate Arena Glass plate G. mellonella larvae Pipette tip Chamber with gradient Nematodes Agar substrate Rubber c) Laboratory results toward non-target hosts: Application point of nematodes The non-insect organisms, earthworms, scorpions and millipedes have been challenged with different strains of entomopathogenic nematodes. Material and methods Hosts: the non-insect organisms, earthworms, scorpions and millipedes have been captured under stones, and stored at 12°C for 24 hours prior the experiment. Nematodes: were reared in Galleria mellonella last instar larvae by standard rearing procedures. For earthworms the following nematode strains were tested: Heterorhabditis bacteriophora NJ, Steinernema kraussei N0093 and S. feltiae UK. For Scorpions and Millipedes: H. bacteriophora NJ, S. kraussei N0093, S. sp. N0166, S. sp. N0167, S. sp. N0168, H. sp. N0169, H. sp. N0170. The infectivity of nematodes was tested in parallel on 100 mg larvae of Tenebrio molitor (10 larvae per box with 1500 nematodes). There were 3 repetitions per treatment. Incubation was done at 23°C for 5 days. Controls (only water) were always included. Tests with Earthworms: in 10x7x5 cm plastic boxes, 50 g of peat with 75% humidity, 5 earthworm were challenged with 3000 infective nematodes. There were 3 repetitions per treatment. Incubation was done at 23°C for 7 and 14 days. Tests with Millipedes: in 10x7x5 cm plastic boxes, 50 g of peat with 75% humidity, 3 millipedes were challenged with 1500 infective nematodes. There were 3 repetitions per treatment. Incubation was done at 23°C for 5 days. Tests with Scorpions: they were tested in Petri dishes with 3000 nematodes per individual for 5 days at 23°C. Results Mortality of Earthworms challenged with entomopathogenic nematodes (see Tab. 1) did not differed among treatments both at 7 (P=05957) and 14 days (P=0,7520). 129 Plexiglas lid PDF creato con FinePrint pdfFactory versione dimostrativa http://www.secom.re.it/fineprint
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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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Il settore oggi è regolamentato in
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target botanical synthetic sodium c
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RELIABILITY AND PROSPECTIVES FOR TH
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Vineyard Passive diffusion Spontane
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of the host, a relatively limited n
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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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5. Hohler, D. (1998). Ochratoxin A
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Table 3: Pesticide treatments on
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BIOPESTICIDES: EVALUATION PROCESS F
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Xenorhabdus spp. and Photorhabdus s
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It can be reproduced in vivo at 15
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Vinson, S. B., 1975, Biochemical co
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PROTECTION OF GRAPEVINE FROM POWDER
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I MEZZI TECNICI PER L'AGRICOLTURA B
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POSTER 1. Agosteo G.E., Ambrosio M.
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31. Pietri A., Bertuzzi T., Barbier
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1 PROVE DI LOTTA ALL’OIDIO DELLO
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2 LUPINUS ALBUS, AN ANCIENT EUROPEA
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Table 1. Benzo(a)pyrene and benzo(e
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5 INSETTICIDI D’ORIGINE VEGETALE
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Tabella 2. Piretrine: composizione
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californicus (fitoseide predatore)
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7 NATURAL COMPOUNDS IN THE CONTROL
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8 METHODS FOR EVALUATION, IN CONTRO
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Figure 3. Effect of Pseudomonas sp.
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9 IMPROVEMENT OF SOIL PROPERTIES AN
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(mg kg-1) (mg kg-1) 140 120 100 80
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10 NEMATICIDAL ACTIVITY OF AQUEOUS
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% cumulative hatch 40 35 30 25 20 1
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ROTENONE Il principio attivo è est
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- azadiractina nei confronti di F.
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