and fate of pesticidal proteins from Bacillus thuringiensis spp in
tropical soils and waters as influenced by clays
The microbe Bacillus thuringiensis (Bt) produces a crystal which
is commonly used to control the presence of insect larvae on crops. Preliminary
studies have shown that Bt toxins (subsp. kurstaki, that protects
against a range of butterfly and moth larvae) can bind on clays. Such
binding reduces the biodegradation of the toxins but does not eliminate
their toxicity to insect larvae. However, few studies on the persistence
and toxicity of Bt toxins in soils and sediments have been conducted.
We have chosen to study the interaction of Bt toxins with mineral colloids
such as reference clays, soil-derived-clays, and natural soils, and to
study the effects of such surface interactions on the persistence and
pesticidal activity of these toxins in soils and waters. This study will
allow us to evaluate any potential effects on tropical soils and waters
of toxins produced by genetically modified organisms that contain genes
that code for these pesticidal toxins (i.e., transgenic plants and micro-organisms).
Approach and methodology
We prepared Bt toxin/colloidal clay complexes by equilibrium adsorption
and binding of the toxins onto test clays. Larvae of insects from the
tropics (pests and non-pests) were used in bioassays to assess the toxicity
of the free toxins and complexes.
the bioavailability of free and bound toxins, we investigated microbial
degradation using inocula from tropical soils. CO2 evolution, enzyme activities
and species diversity were measured to evaluate the effects of Bt toxins
on the activity, ecology and population dynamics of soil microbiota.
and binding assays were performed on soils and their clay fractions to
evaluate the persistence and effects of the toxins.
Main findings and outcome
A multilinear regression equation was found to account for the adsorption
of true toxin (63 kDa) of Bt tenebrionis on a reference clay (Na-montmorilonite).
The distribution of the toxin between the bulk solution and the clay surface
appears to be regulated by the ionic strength of the medium and to a lesser
extent by its pH. The toxin has two forms: that adsorbed from the surrounding
water solution at very low ionic strength and the additional amount adsorbed
at different ionic strengths. The first fraction is not easily desorbed
by water alone and can be considered as irreversibly bound. This bound
form is not toxic in bioassays with larvae of Leptinotarsa decemlineata
(Colorado potato beetle). The second form can be easily desorbed when
the ionic strength is lowered and has been shown to be very active.
the ionic strength and pH in the midgut of larvae may change and desorb
the toxin from the clay. Additionally, the adsorbed toxin can safely reach
the receptors, possibly because it may escape proteolysis in the very
first part of larvae's alimentary canal. Experiments on soils and clay
from soils seem to confirm these findings.
address whether the Bt toxins produced by GMOs can accumulate in soils.
The model, with some corrections/ adjustments could be used as a forecasting
tool to monitor the behaviour of toxins in soil environments. Early results
of microbiological assays indicate that the microbial populations (bacteria,
fungi, and actynomicetes) are not quantitatively or qualitatively influenced.
Bioassays on non-target Coleoptera species, which spend part of their
life cycle in the soil, indicate that the purified Bt tenebrionis toxin
(63 kDa) is not effective against earthworms (Eisenia foetida)
when mixed with soil. Similar studies will be performed on true toxin
produced by Bt kurstaki, as this toxin is often present in transgenic
crops such as corn.
The improvements in the potency and persistence of Bt formulations, as
well as the engineering of transgenic organisms able to express the introduced
Bt genes, have created the potential problem of the accumulation of Bt
toxins in the environment, especially in the residues of transgenic organisms
(particularly plants). This could be harmful to non-target species and
could result in the selection of toxin-resistant target species.
of the toxins is particularly important in soils and sediments where the
toxins can bind on colloidal particles (e.g., clay minerals) and, thereby,
be rendered less accessible to microbial degradation. The model developed
for Bt tenebrionis is a starting point to predict the behaviour
of this toxin in soil environments. Moreover, it can account for the high
toxicity of adsorbed toxin. Bt toxin was shown to have negligible effects
on soil microbiota and non-target organisms such as earthworms.
Gonzalez P., Fereres A., Risk assessment of Bt toxins adsorbed
or bound to clay minerals.
Phytoparasitica, 27 (4), 1999, p. 301.
Muchaonyerwa P., Chenu C. and Pantani O.L., Devenir dans les
sols de la protéine insecticide de Bacillus thuringiensis
var tenebrionis: adsorption sur les fractions argileuses de sols
tropicaux, Poster presentation by C. Chenu, Sixièmes
journées Nationales de LEtude des Sols, Nancy, 25-28
Muchaonyerwa P., Chenu C., Pantani O.L. and Calamai L., Adsorption
of toxin from Bacillus thuringiensis var. tenebrionis to clay fractions
from tropical soils. Accepted, Oral presentation, ISMOM 2000
Soil Mineral - Organic Matter - Micro-organisms Interactions in
Ecosystem Health, Naples, 22-26 May 2000.
González P., Pantani O.L., Ristori G.G. and Fereres A., Activity
and persistence of Bacillus thuringiensis subsp. israelensis crystals
mixed with clays against the mosquito Culex pipiens, Oral presentation,
ISMOM 2000 Soil Mineral - Organic Matter Micro-organisms
Interactions in Ecosystem Health, Naples, 22-26 May 2000.
Pantani O.L. and Calamai L., Sorption of the toxin from Bacillus
thuringiensis subsp. tenebrionis on na-montmorillonite, as influenced
by ph and ionic strength, may also affect its bioavailability,
Poster presentation, ISMOM 2000 Soil Mineral - Organic Matter
Micro-organisms Interactions in Ecosystem Health, Naples, 22-26
March 1998 February 2001
Istituto per la Genesi e l'Ecologia del Suolo IGES
University of Fort Hare ALICE (ZA)
University of Zimbabwe (ZW)
S. Madoffe Sokoine
University of Agriculture