International Congress BIOLOGICAL PRODUCTS - Gruppo di ...

Xenorhabdus spp. and Photorhabdus spp. are carried in the intestine of the infective stages of nematodes; Steinernema
spp. have a special intestinal vesicle where the bacteria are kept while Heterorhabditis spp. have their symbionts in the
lumen of the anterior part of the gut.
Once the nematodes have penetrated a host, they release the bacteria symbiont in the haemolymph causing a
septicaemia and the host dies. Inside the larval cadavers the bacteria growth and nematodes develop to adult stages,
multiply and sexually reproduce. Although the nematodes can grow axenically (without any micro-organism) or on
other bacteria, their reproduction is optimal only in presence of their natural symbiont. Thus, Xenorhabdus spp. and
Photorhabdus spp. have the status of obligate symbiont because they provide essential nutrients for the correct
multiplication of the nematodes (Boemare et al., 1997).
The nematode-bacterium complex represents an exciting example of co-evolution that involves the interaction between
the host and the nematode-bacterium complex, the relationship within the two symbiotic partners and the interaction
between the bacterium and its bacteriocines. To investigate these three connections, it is necessary to study the insect
defence reactions, the pathogenicity of the nematodes and the bacterium pathogenicity, symbiotic properties and
lysogeny.
Insects have an immune system based on phagocytosis and encapsulation of exogenous agents. The recognition of
foreign bodies is mediated by the production of some humoral factors (Boemare et al., 1997). Entomopahtogenic
nematodes are able to depress or to escape the immune defence of insect larvae. This is obtained both with nonrecognition
by insect humoral factors and by escaping the phagocytosis and encapsulation. The nematodes secrete an
immune-depressive factor against insect immuno-proteins directed against the symbiotic bacteria. As been reported also
that axenic nematodes produce toxin(s) able to cause paralysis and death of the host (Simoes, 1994). Symbiotic bacteria
display also pathogenic action them-self: they produce toxins and secrete enzymatic complex (proteases, lipases and
phospholipases) that help the establishment of septicaemia in the host. Thus both partners of the symbiotic complex
collaborate to kill the host. It is also been reported that axenic S. glaseri or its sole symbiont X. poinarii, have no
entomopathogenic action, but this was restored after re-association of both partners (Akhurst and Boemare, 1990). The
observed lysogenity of the bacterial symbiont is also useful for the symbiosis relationship because it helps the
symbionts to compete with closely related bacteria.
Production of useful molecules
Both Xenorhabdus and Photorhabdus spp. can be grown independently from they nematodes partners under standard
laboratory conditions. In vitro, bacteria secrete several extracelluar products: proteases, chitinases, lipases,
phospholipases, antibacterial and antimycotic substances. Those compounds are produced also in vivo and the enzymes
digest the tissues of dead larvae in order to provide nutrients for both bacteria and nematodes. The antifungal and
antibacterial products are used to preserve the cadavers from the colonisation of other microorganisms. Those active
substances have also been tested for their use in biolgical control of phytopatogenic fungi (Ng and Webster, 1997).
In vitro, X. nematophilus and X. bovienii produce aliphatic amides (Park and Paik, 2001) and dithiolpyrrolones tested
for antineoplastic activity (Webster and Chen, 1999).
Recently, strains of Photorhabdus capable to secrete exotoxins (Bowen et al., 1998), or to produce endotoxins (Ragni et
al., 1997), with oral insecticidal activity, have been described. This new source of natural toxins could help both the
fight against the development of insect resistance as well as novel sources of specific activities in combination with
other known bio-pesticides.
Target organisms
Due to their poor resistance to dry conditions, EPNs are mainly applied against soil dwelling insects. Otiorrhynchus
sulcatus F. (Coleoptera, Curculionidae) is considered one of the major worldwide insect pests. The larvae feed on the
root system of several plant species (mainly ornamentals and nursery stock) producing its consequent decay and very
significant economic losses. A part other Curculionids such as Conorhychus mendicus, Balaninus elephas, Diaprepes
abbreviatus and Cylas formicarius on ornamentals, berries, citrus, sugar beet and chestnuts, other pests are: Scarabeids:
Popilia japonica, Maladera matrida, Phyllopertha horticola , Aphodius spp. on lawns, orchards, nurseries and sweet
potatoes; Diptera: Sciarids, Phorids, Scatopsids, Cecidomids on mushrooms, greenhouses; Lepidoptera: Noctuids on
vegetable crops. Several studies have been performed to find out if EPNs can be used on the aerial part of the plants
against chewing and sucking insect, but, yet, without any important success.
Application Technology
Particular strategies must be developed in order to insure the successful delivery of the nematodes to the target site and
target insect. Many parameters must be investigated to improve EPNs performance. One of these is the determination of
target insect life-stage susceptibility, since different life stages of different species are not equally susceptible. It has
been shown that pest population levels and behaviour have a great influence on nematode performance and must be
considered carefully. Often, larval stages of insects such as borers are not accessible to nematodes. Selecting the most
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