International Congress BIOLOGICAL PRODUCTS - Gruppo di ...

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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
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