The Endophytism of Bacillus thuringiensis in
Cotton Plants at Acquisition and Oviposition
by Bemisia tabaci
Flávia Santana S da Costa1*, Marcelo Tavares de Castro2 and Rose Monnerat3*
1 University of Brasília, Brazil
2 University Center ICESP, Brazil
3 Empresa Brasileira de Pesquisa Agropecuária (Embrapa) – Embrapa Genetic Resources and Biotechnology (Embrapa Cenargen), Brazil
Submission: November 12, 2021; Published: November 19, 2021
*Corresponding author: Flávia Santana S. da Costa, University of Brasília, University Campus Darcy Ribeiro - CEP 70910-900 Brasília, Brazil - E-mail: firstname.lastname@example.org
Rose Monnerat, Empresa Brasileira de Pesquisa Agropecuária (Embrapa) - Embrapa Genetic Resources and Biotechnology (Embrapa Cenargen), Parque Estação Biológica, PqEB, Av. W5 Norte (final). Caixa Postal 03372, CEP 70770-917 Brasília, Brazil - E-mail: email@example.com
How to cite this article: Flávia S S d C, Marcelo T de C, Rose M. The Endophytism of Bacillus thuringiensis in Cotton Plants at Acquisition and Oviposition
by Bemisia tabaci. Agri Res & Tech: Open Access J. 2021; 26 (3): 556340. DOI: 10.19080/ARTOAJ.2021.26.556340
Beneficial microorganisms can be applied on the soil or seeds and can protect plants against insect pests or pathogens. It is known which Bacillus thuringiensis (Bt) strains that, when introduced into the soil can be absorbed by the plant and translocate throughout this with insecticidal activity against insect pests that feed on them. For this reason, the ability of Bemisia tabaci ‘B biotype’ (Hemiptera: Aleyrodidae) to ingest Bt sap was evaluated in cotton plants inoculated with the Bt strain, which encodes the GFP gene, Btk-gfp (B. thuringiensis subsp. kurstaki - green fluorescent protein), and tested for oviposition, comparing untreated plants to those treated with Bt strains. The recovery of Btk-gfp was observed in the plant along with its ability to be acquired by nymphs and adults of B. tabaci. Plants treated with Bt strain S1806 had a lower number of B. tabaci eggs than plants that received only sterile water.
Abbreviations: Embrapa: Empresa Brasileira de Pesquisa Agropecuária; Embrapa Cenargen – Embrapa Genetic Resources and Biotechnology; Bt: Bacillus thuringiensis; Btk-gfp: B. thuringiensis subsp. kurstaki: green fluorescent protein; GFP: Green Fluorescent Protein; B. tabaci: Bemisia tabaci; AbMV: Abutilon Mosaic Virus; P. xylostella: Plutella xylostella; UV: ultraviolet; ISLA: Brazil seed production company; BRS 8H: variety of cotton plants developed at Embrapa; rpm: revolution por minute; M: meters; μL: microliter; μg: microgram; nm: nanometer; h: hours; mL: milliliter; °C: degrees Celsius; ANOVA: Analysis of variance; F: test in ANOVA; p: value statistical significance
The cotton plant (Gossypium hirsutum L., Malvaceae) production in Brazil attracts and hosts a significant complex of pests that attack the roots, stem, leaves, flower buds, fruits, and bolls . Pests can cause significant yield losses in addition to raising concerns about sustainability of the productive system. Pest control in cotton is expensive and primarily done with broad-spectrum synthetic chemical insecticides . To meet the growing demand for fibers, the crop must be protected from pests while still preserving natural resources, maintaining environmental quality  and ensuring human health. Thus, research continues to look for new measures to control these problems.
The whitefly [Bemisia tabaci (Gennadius) ‘B biotype’ (Hemiptera: Aleyrodidae)] is an important pest of cotton crops [4,5]. The damage caused by B. tabaci ‘B biotype’ includes,primarily, the large extraction of sap and, indirectly, the transmission of viruses, including the “common mosaic” AbMV (Abutilon Mosaic Virus) . This biotype had shown to have a great capacity to develop resistance to most of the active ingredients on the market, including neonicotinoids and growth regulators [7-9] that also directly affect the natural enemies . Through the excessive feeding of phloem sap, the honeydew excreted from B. tabaci support the growth of sooty mold fungi (Capnodium sp.), that inhibits host photosynthesis  damaging the processing and depreciating the fiber due to the lack of the product standardization [12,13]. The wide distribution of the whitefly is also related to the agricultural production system, such as the succession and sowing of host crops, in addition to wild species that contribute to the continuous population growth of B. tabaci as well.
Biopesticides containing organisms entomopathogens are
environmentally friendly and often decompose quickly, resulting in
lower exposures and avoiding the population problems caused by
synthetic chemical pesticides . Bacillus thuringiensis Berliner
(Bacillaces: Bacillaceae) (Bt), is an entomopathogenic bacterium,
which has been widely used worldwide in spray able biopesticides
formulations and in transgenic Bt-plants. The spores and the
toxins crystals used in formulations provide good protection of
plants crops against attack by insect pest, and control of insect
species of the orders Lepidoptera, Diptera, Coleoptera . Some
of isolates are also active against other insect orders such as
Hemiptera, Hymenoptera, Homoptera and Orthoptera [16-19].
The use of strains and derived products of Bt has been
previously proposed as a feasible alternative to manage B. tabaci
[8,20-23] in bioassays realized with Bt isolates and toxins that
were exposed on the leaves surface and on insect bodies. However,
no commercial bioinsecticide have been produced using this
bacterial to control B. tabaci so far . Among these reports, there
are few studies that investigated endophytic Bt against sucking
pests  and none with B. tabaci. Studies indicate the possible
success of this technology demonstrating that Bt colonizes cotton
and cabbage tissues and, therefore, could be available to insects
that feed on them, such as Spodoptera frugiperda (JE Smith)
(Lepidoptera: Noctuidae) and Plutella xylostella (L.) (Lepidoptera:
Plutellidae), respectively . The use this Bt technology can help
control P. xylostella in cabbage plants and achieved control of
Hypsipyla grandella Zeller (Lepidoptera: Pyralidae) in mahogany
seedlings [26,27]. There is a close relationship between the
Bt strain and the cotton variety, and that Bt can colonize plants
endophytically, acting simultaneously to promote plant growth
and potentially control insects . The endophytic form of Bt can
be an alternative that could decrease the problem of the bacteria’s
sensitivity to ultraviolet (UV) light and rainwater.
For this reason, the aim of this work was to evaluate the
capacity to recovery Bt from nymphs and adults of B. tabaci ‘B
byotipe’ fed on cotton plants inoculated with the Btk-gfp (B.
thuringiensis subsp. kurstaki: green fluorescent protein) strain,
which encodes the GFP gene. In this way, to verify the ability of this
sucking pests to acquire sap Bt. Other objective was to evaluate
the oviposition comparing untreated plants to those treated with
The adult whiteflies were obtained from a stock colony
maintained on cabbage plants Brassica oleracea var. acephala
(ISLA Seeds, Rio Grande do Sul State, Brazil). Eight plants were
kept in cages built with PVC pipes and voile type fabric in
greenhouse conditions at (28±2)°C and (60±10) % humidity. The
plants were irrigated and replaced as needed to maintain their
To prepare the plant material, 4 pots in polyethylene
(dimensions 20×32cm), containing 3 cotton plants (variety BRS
8H) at 18 days after sowing, were filled with 500g of sterile soil
and BioPlant® (Minas Gerais, Brazil) substrate in 2:1 ratio. The
microorganism strain, which encodes the GFP gene, Btk-gfp (B.
thuringiensis subsp. kurstaki: green fluorescent protein) was
grown in liquid medium supplemented with erythromycin (10μg
mL-1) and incubating the culture for 48h., 200rpm at 28°C .
Then 5mL of the obtained bacterial suspension was inoculated
into the soil close to the root of each plant. Two pots were used to
inoculate the bacteria containing the fluorescent protein and the
negative control using sterile water.
Five days after the inoculation, 35 non-sexed adult B. tabaci
were confined to cotton plants in a cage made with voile fabric and
a wire ring for 48hours to permit feeding and oviposition. The soil
was covered to avoid contact with flies. After this period, the adults
were collected with the aid of an insect aspirator, and the plants
were transferred to (0.3×0.8×0.3m) cages also covered in voile
to observe the Btk-gfp ingestion by B. tabaci nymphs (about 80
individuals collected from cotyledon leaves and the first true leaf
node) that occurred 11 days after the end of the adult exposure.
The insects’ adults were superficially disinfected with 70%
alcohol, 2% hypochlorite, and sterile distilled water for 10seconds
in each solution and powdered in the presence of 0.9% NaCl. An
aliquot of 25μL was seeded in triplicate in Petri dishes containing
Embrapa selective agar medium (erythromycin at a concentration
of 10 μg/mL) and incubated at 28°C for 48h at 200rpm .
After this period with Drigalski (1μL) cell spreader, a portion of
this bacterium was collected, and its colonies were visualized
in the dark with fluorescence microscopy in a Zeiss Axiophot
(Jena, Germany) microscope using a 475-550nm filter, to detect
the presence the GFP in vegetative cells. The same process was
conducted for nymphs, but without disinfestation.
The Bt strains used in this bioassay were randomly selected
with different serotypes and strains tested to promote cotton plant
growth and against S. frugiperda , and in cabbage seedlings
. Ten strains were tested: S456 (B. thuringiensis subsp.
entomocidous), S546 (B. thuringiensis subsp. kurstaki), S599 (B.
thuringiensis subsp. kenyae), S655 (B. thuringiensis subsp. alesti),
S907 (subsp. not determined), S1166 (B. thuringiensis subsp.
muju), S1450 (B. thuringiensis subsp. kurstaki HD1 (Btk)), S1806
(B. thuringiensis subsp. japonensis), S1905 (B. thuringiensis subsp.
kurstaki), and S2122 (B. thuringiensis subsp. kurstaki). The strain
S1450 HD1 (Btk) obtained from the Collection of B. thuringiensis
and Lysinibacillus sphaericus at the Pasteur Institute, France, and
the others strains from the Bank of Bacteria of Invertebrates at
Embrapa Genetic Resources and Biotechnology, located in Brasília,Federal District at Brazil.
For the oviposition bioassay, a new cage was assembled like
the breeding cage and under the conditions described above, now
containing six cabbage plants infested with B. tabaci adults. The
cotton plants were placed in pots (80mL) containing a mixture
of sterile soil and Bioplant® substrate in a 2:1ratio. After the 15-
day period of emergence, the plants were ready for testing. The
inoculum was prepared by adding a strip of filter paper containing
spores (stored in the bacteria bank) to 15mL of Embrapa medium
and incubating the culture for 72h., 200rpm, 28°C . The growth
was observed by a phase contrast optical microscope system to
verify the bacterium structures in the formation of spores and
The cotton plants were inoculated with 5mL of this bacterial
suspension onto the soil close to the roots, and the plants were
allowed to sit for 48h before exposure to insects. The plants with
bacteria were placed in rows in the cage for the bioassay so they
were between the whitefly-infested plants. The system remained
for a period of 12hours for oviposition, when the plants were carefully removed from the cages to count the eggs and examined
microscopically with the aid of a stereoscopic magnifying glass
(Leica Microsystems, Wetzlar, Germany) at x 40 magnification.
The control treatment consisted of plants treated only with
sterile water and plants inoculated with Embrapa medium
without the bacteria. For each strain was prepared five replicates
in a completely randomized design. The data were submitted to
ANOVA variance test and Tukey test (p<0.05) using the Sisvar
The ability to acquire and recovery Btk-gfp by B. tabaci was
tested. Bacterial growth was obtained from macerated B. tabaci
adults and nymphs that had been fed on plants treated with Btkgfp
(Figures 1 & 2). The bacterial growth obtained from adult and
nymphs was observed with epifluorescent lighting and showed
cells that express green fluorescent protein. Plant roots probably
absorb Bt bacteria, making them available for food in adult hood
and immature whitefly. The insects collected from control plants
did not display bacterial growth.
In the test with different serotypes and Bt strains the
oviposition of B. tabaci on inoculated cotton plants was evaluated.
After 12 hours of infestation, interesting results between
treatments were observed (Figure 3). The plants with strain
S1806 exhibited a lower number of B. tabaci eggs compared
to the control with only water, and strains S456 and S1166 (F =
1.794; p = 0.08145). However, this strain was similar with other
tested isolates and the control with the culture medium used to inoculate the roots of the plants. Plants containing inoculum with
strains S1166, S1450, and S655 showed a greater egg density with
511.4; 453.0, and 336.6 eggs per plant, respectively. The least eggs
were noted for S1806 with 108.8 eggs and S907 with 197.6 eggs
per plant. The treatment containing strains S599, S1905, S2122,
S456, S546 and the control with Embrapa medium varied between
223.8 and 296.2 eggs per plant
Bemisia tabaci ‘B biotype’ is an important pest of cotton crops
[4,5] wich has developed resistance to the chemical insecticides
[8,9] affecting the agricultural economy . An alternative
could be the use of B. thuringiensis, but until now, there has not
been commercial products containing these bacteria to B. tabaci.
Bacillus thuringiensis insecticidal proteins are used as active
components of biopesticides and as protectors incorporated into
the plant in transgenic crops. One of the most relevant attributes
of these Bt protein-based insecticidal technologies is their high
specificity, which guarantees the absence of harmful effects on
non-target insects, vertebrates, and the environment .
The use of endophytic organisms can bring benefits in
biological control of plant pathogens and insect-pest [32-34]. The
adoption of this technology has been investigated using Bt with
relevant control results on important pest in agriculture [24-28],
but no study verified this effect on B. tabaci. In this work, Btkgfp
recovery and acquisition capability of B. tabaci fed in cotton
plants the sap Bt was checked. The collected cells from the bodies
of adults and immature stages of the whitefly produced bacterial
colonies morphologically similar to Bt after maceration, plating
and visualization through optical fluorescence microscopy.
This practice was used with efficiently to confirmation of the
colonization of Bt strains in plants and to demonstrate the toxic action of Bt against the sucking pest Aphis gossypii Glover
(Hemiptera: Aphididae) .
Another study demonstrated the ability of Bt strains to
translocate from roots to shoots in citrus seedlings, with capacity
of use in control of the phloem-feeding insects Diaphorina citri
Kuwayama (Hemiptera: Lividae) that feed on the plant’s shoots
. In summation, the inoculated Bt had been absorbed by the
roots and transported to the aerial part of the plant by the xylem
vessels [24,25,28], but the mechanisms of transport are not yet
elucidated. This work suggests that this transport could also occur
for the plant phloem, which is the preferred feeding place for B.
tabaci, even though it often tastes the xylem vessels .
Some reports have demonstrated toxicity activity of the Bt
strains or toxins to hemipterans. Nymphs of whitefly treated
directly with the suspension containing the complex crystal-spore
of Bt showed a 90% major mortality . In that research, the strain
GP139 of Bt was used to show the toxic activity against B. tabaci
, in addition the high virulence of this Bt strain observed in
bioassays performed with Myzus persicae Sulzer (Hemiptera:
Aphididae) . Bacillus thuringiensis isolates were selected for
their high insecticidal potential against nymph’s whitefly under in
vitro conditions . Native isolates of Bt from soil environments
caused up 50 to 70% mortality of B. tabaci nymphs  and
mortality with superior percentage of 90% . Consistent effects were found from isolates and supernatants derived products of Bt
culture on adult repellence, oviposition deterrence and nymphal
mortality of B. tabaci .
In these reports, the studies evaluated strains, toxins and
derived products of Bt against B. tabaci by immersion or spraying
of the bacterial suspension in surface of leaves and insect bodies
[8,20,22,30,37,38]. But the action of Bt sap has not been reported
to this pest.
The oviposition test comparing untreated cotton plants to
those treated with serotypes and Bt strains, showed a lower eggs
number to the S1806 strain. It is supposed that the colonizing
capacity of Bt inside the cotton plant can interfere in the oviposition
of the whitefly, which feeds on those structures, with potentially
insecticide activity. Determining which mechanisms were
responsible for the difference in oviposition is still premature.
However, herbivores can use plant volatiles to locate their hosts
, and cotton plants treated with growth-promoting bacteria
can modify the volatile profile.
The effects of the volatiles on plants treated with the beneficial
bacteria can reduce the oviposition of pest insects . Females
during their search for oviposition lay their eggs on healthier plants
for better-quality larval development. Plants treated with Bacillus
subtilis showed low populations of whitefly in greenhouse .
Another research group reported that the treatment of bacteria in
tomato plants reduced the emergence of whitefly adults and that
this suppression was due to jasmonic acid responses  in plantinduced
systemic resistance. Many studies have reported the use
of Bt as an insecticidal product or the insertion possibility of its
toxins into plants to provide resistance to susceptible insects.
However, little is known about how plant-colonizing bacteria
act against harmful insects that feed on them. Reports discuss
metabolically active form of Bt in the phylloplane .
The resistance induced by microorganisms to insects varies
between studies, due to different hosts, different plants, insects,
and microbial inoculants [44,45]. Therefore, different responses
may be found for insect pests interacting with the plant treated
by the bacterium. The effects of treatment with bacteria on insect
pests may also vary in relation to insect eating habits, whether it
is a specialist or a generalist. Beneficial bacteria that have some
effect in promoting plant growth appears to negatively affect
the development of insect pests [24,26,28,32]. The recovery
capacity of Bt by adult and immature B. tabaci demonstrates the
possibility of exploring the Bt sap as an alternative biological
control technology for this insect, in an endophytic way in the
plant. The result obtained opens new perspectives for studying
the interaction of endophytic Bt in plants for biological control of
sucking pests of great agronomic interest.
Bemisia tabaci ‘B biotype’ are able to acquire Bt inoculated in
cotton plants by feeding on their structures. Plants treated with Bt strain S1806 had a lower number of B. tabaci eggs than plants that received only sterile water.
The authors gratefully thank the Coordination of Improvement
of Higher Education Personnel (CAPES, in Portuguese) for
financial support received by first author and to Dr. Francisco
Aragão (Embrapa Cenargen) for your help in starting the whitefly
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Rodrigues SMM, Vivan LM (2007) A Mosca Branca (Bemisia tabaci Biótipo B) no Mato Grosso. Embrapa, Circular Técnica 111 (CNPA) (infoteca-e), Embrapa Algodão, Campina Grande, Paraíba, Brazil p 9.
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