International Congress BIOLOGICAL PRODUCTS - Gruppo di ...

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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.
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