Contents - Faperta

Genetic Engineering of Entomopathogenic Microbes for Pest Management 271
(Lampel et al., 1994), and the resulting recombinant strain has been used to inoculate corn
for the control of European corn borer, Ostrinia nubilalis (Hubner) infestation (Tomasino
et al., 1995). Endophytic isolates of B. cereus have been used as hosts for the cry2Aa gene
(Mahaffee, Moar, and Kloepper, 1994), and a B. megaterium isolate that persists in the
phyllosphere (Bora et al., 1994) has been used as a host for cry1A genes. Similarly, cry genes
have been transferred into other plant colonizers, including Azospirillum spp., R. leguminosarum,
P. cepacia, and P. fl uorescens (Obukowicz et al., 1986a, 1986b; Skot et al., 1990; Stock
et al., 1990; Udayasurian et al., 1995). Nambiar, Ma, and Iyer (1990) expressed toxin gene
from B. thuringiensis subsp. israelensis into Bradyrhizobium species that fi x nitrogen in
nodules of pigeonpea. The plasmid was transferred by conjugative mobilization into a
Bradyrhizobium species that nodulates pigeonpea. Experiments in a greenhouse indicated
that this provided protection against root nodule damage by larvae of Rivellia angulata
(Hendel). Alternative delivery systems have also been sought for the dipteran-active toxins
of B. thuringiensis subsp. israelensis to increase their persistence in aquatic systems. Such
hosts include B. sphaericus (Bar et al., 1991; Poncet et al., 1994), Caulobacter crescentus
Poindaxter (Thanabalu et al., 1992), and the cya nobacteria, Agmenellum quadruplicatum
(Menegh.) Brebisson. (Stevens et al., 1994) and Synecho coccus spp. (Soltes-Rak et al., 1993).
The gene encoding a 65 kDa polypeptide toxin from B. thuringiensis subsp. tenebrionis,
which is lethal to coleopteran insects, has been cloned in E. coli in the broad host range
vector pKT230 and subsequently transferred to R. leguminosarum by conjugation, producing
two major polypeptides of 73 and 68 kDa (Skot et al., 1990). Cell extracts from toxinproducing
rhizobia were toxic to larvae of Gastrophysa viridula (Deg.). Bioassays also
showed that the delta-endotoxin was toxic to larvae of Sitona lepidus Gyllen. Pea and white
clover plants suffered lower root and nodule damage by larvae of S. lepidus when inoculated
with Rhizobium strains containing the toxin gene.
Cyanobacteria, Agmellenum sp., Snechococcus sp., and C. croscentus have also been used to
deliver the Bt toxins in aquatic environments (Thanabalu et al., 1992; Soltes-Rak et al., 1993;
Stevens et al., 1994). Other species of bacteria have been used on a much smaller scale in
pest control. The cryIAc gene has been introduced into the chromosome of P. fl uorescens
isolate 14 using an artifi cial transposon- carrying vector, Omegon-Km. Bioassays on Eldana
saccharina Walker larvae have shown that the strain carrying the gene was as toxic as the
one carrying it on pKT240 (Herrera, Snyman, and Thomson, 1994; Herrera et al., 1997).
Sugarcane treated with P. fl uorescens 14: Omegon-Km-cry suffered lower stem borer damage
than the untreated sugarcane. A transgenic strain of P. fl uorescens containing the cry1Ac
gene from a wild-type strain of Bt has been evaluated for its ability to colonize the external
surface of sugarcane (Black, Huckett, and Botha, 1995). Plants inoculated with nontransgenic
and transgenic strains of P. fl uorescens showed that viable cells were recoverable for
three months, although cry1Ac gene was detectable by polymerase chain reaction (PCR)
for up to eight months. Material treated with the combination innoculum showed that the
nontransgenic P. fl uorescens strain outnumbered the transgenic counterpart. When the
cryIAc gene was integrated into P. fl uorescens P303-1 by electroporation (Duan, Zhang, and
Xu, 2002; Duan et al., 2002), the engineered bacteria were highly insecticidal to cotton
bollworm, H. armigera. The LC 50 against the second instars after fi ve days was 50.13 to
192.87 μg g 1 . Mortality of the treated larvae showed a positive correlation, while weight
showed a negative correlation with the bacteria concentration. In vitro bioassays with
second-instar H. armigera showed that the engineered strains PT45, PT51, PT61, and PT71
had greater insecticidal activity than Bt strain HD-73 (Duan, Zhang, and Xu, 2002). Larvae
fed on leaves suffered 70.0, 60.0, 60.0, 66.7, and 33.3% mortality, respectively. Larval weight
showed a negative correlation with concentration.