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30 ISOLATION OF NEW BIOLOGICAL CONTROL AGENTS FROM UMBRIA REGION M. Scribano and A. Ragni BioTecnologie BT srl Pantalla di Todi 06050 Perugia Italy Tel: ++39 – 075 - 895091 Fax: ++39 – 075 – 888 776 E-mail: aragni.bt@parco3a.org Summary Samples of soil were collected in two areas of the Umbria region, Valnerina and Oasi di Alviano; the samples were used for the isolation of entomopathogenic agents through a selective technique based on the usage of bait. There were isolated 13 different samples of entomopathogenic nematodes, 25 entomopathogenic fungi, 27 sporogenous bacteria and 44 non-sporogenous bacteria. The activities of these microbiological agents were verified through in vitro and in vivo tests. Materials and Methods 1. Sampling: 80 soil samples (1 Kg each) were collected till the depth of 20/25 cm from two natural areas of Umbria region: Valnerina and Oasi di Alviano. 2. Baiting with Galleria mellonella 1 : the soil samples were put in containers together with a bait, larvae of the Galleria mellonella insect; they were incubated in the dark at 25°C. Periodically the boxes were controlled; the dead insects were collected and used later for the isolation of microbiological agents. 3. Isolation of nematodes: the collection of nematodes was carried out through White-trap 1 : the gummyconsistence Galleria cadavers were placed on a filter paper imbibed with Ringer solution in order to allow the nematodes to emerge (fig1). The entomopathogenic power of collected nematodes was tested through infections of Galleria mellonella larvae 2 : 7 last-instar larvae of Galleria mellonella were treated with 100 nematodes per larva. 4. Isolation of fungi 3 : the hyphae, developed on the surface of the turgid cadavers, were streaked on PDA (potato dextrose agar) Petri dishes containing 150 µg/ml of streptomycin. Than the dishes were incubated at 25°C. 5. Isolation of bacteria: Non sporogenous bacteria: the flabby cadavers of Galleria were homogenized in a physiological solution; the suspension was plated on NA (nutrient agar) Petri dishes containing 150 µg/ml of nystatin. Than the dishes were incubated at 25°C. Sporogenous bacteria: an aliquot of the insect cadaver suspension was treated for 12 minutes at 78°C and then was streaked on NA. The entomopathogenic power of the isolated bacteria towards Mamestra brassicae (Lepidoptera) was verified though in vivo tests using an overlay technique bioassay 4 : 32 neonates of M. brassicae were treated with the fermented broths of the isolated bacteria. Identification of bacteria was performed with gallery API 50 CHB/E Medium. Results: Only 3 out of 27 of the sporogenous bacteria caused 100% mortality towards Mamestra brassicae. API 50 CHB/E test showed that two of three active bacteria strains were Bacillus thuringiensis species as confirmed by microscopical observation of the sporulating cells (fig.2). B. thuringiensis is the most important microbiological agent for control of pest insects 5 . The third strain is being identified.The entomopathogenic power of the 13 isolated nematode strains were tested through infections of Galleria mellonella; the obtained mortality was high although variable, as illustrated in Table1. Conclusion The study has shown the possibility of isolating samples of micro-organisms useful for the biological control from natural habitats. The study will need more screenings in order to evaluate the entomopathogenic power, the conditions of use and the possibility of production of the discovered micro-organisms for using them in the integrated pest management. Acknowledgements PDF creato con FinePrint pdfFactory versione dimostrativa http://www.secom.re.it/fineprint 140
M. Scribano research work was supported by grant of 3A Parco Tecnologico Agroalimentare dell’Umbria, in the frame of Umbria Region Training programm (European Social Forum found). References 1 .K.V. Deseo Kovacs, L. Rovesti. 1992. Lotta biologica contro i fitofagi; Edagricole 2. Bedding, R.A. & Akhurst, R. J. 1975. A simple technique for the detection of insect parasitic rhabditid nematodes in soil; Poinar, G. O. 1979. In: Nematodes for biological control of insects. CRC Press, Boca Raton, Florida; pp.227. 3 Burge, M. N. 1988. Fungi in biological contol systems. Manchester Univ. Press, Manchester & New York; pp.296; 4 Deseo, K. V. 1991. Batteri entomopatogeni come mezzi di lotta microbiolo gica. Inf. Fitopat. 10, 7-13. 5 Li, R. S., Jarrett, P. & Burges, H. D. 1987.– Importance of spores, crystal and delta endotoxin in the pathogenicity of different varieties of Bacillus thuringiensis in Galleria Mellonella. J. Invert. Pathol. 50; 277- 284. Figure 1. White-trap with entomopathogenic nematode parasitized insects PDF creato con FinePrint pdfFactory versione dimostrativa http://www.secom.re.it/fineprint 141
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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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Recovery and repeatabilty tests wer
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13 IMPIEGO DEL TIMOLO NEL CONTROLLO
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dello standard esterno. E’ stata
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Tab. III - Residui di timolo(mg/Kg)
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phytotoxicity index (%) Results In
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15 ORGANIC WHEAT QUALITY AND PRODUC
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