Control of Diatraea saccharalis by the endophytic Pantoea agglomerans 33.1 expressing cry1Ac7

Arch Microbiol (2014) 196:227–234 DOI 10.1007/s00203-014-0962-6

Original Paper

Control of Diatraea saccharalis by the endophytic Pantoea agglomerans 33.1 expressing cry1Ac7 M. C. Quecine · W. L. Araújo · S. Tsui · J. R. P. Parra · J. L. Azevedo · A. A. Pizzirani‑Kleiner 

Received: 13 July 2013 / Revised: 16 December 2013 / Accepted: 4 February 2014 / Published online: 16 February 2014 © Springer-Verlag Berlin Heidelberg 2014

Abstract  Despite the fact that Bacillus thuringiensis (Bt) is found in more than 90 % of the products used against insects, it has some difficulty reaching the internal regions where the larvae feed. To solve this problem, many genetically modified microorganisms that colonize the same pests have been developed. Thus, the endophytic bacterium Pantoea agglomerans (33.1), which has been recently described as a promising sugarcane growth promoter, was genetically modified with the pJTT vector (which carries the gene cry1Ac7) to control the sugarcane borer, Diatraea saccharalis. Firstly, the bioassays for D. saccharalis control by 33.1:pJTT were conducted with an artificial diet. A new in vivo methodology was also developed, which confirmed the partial control of larvae by 33.1:pJTT. The 33.1:pJTT strain was inoculated into sugarcane stalks containing the D. saccharalis larvae. In the sugarcane stalks, 33.1:pJTT was able to increase the mortality of D. saccharalis larvae, Communicated by Erko Stackebrandt. M. C. Quecine (*) · S. Tsui · J. L. Azevedo · A. A. Pizzirani‑Kleiner  Department of Genetics, Escola Superior de Agricultura “Luiz de Queiroz”, University of São Paulo, Av. Pádua Dias 11, P.O. BOX 83, Piracicaba, SP 13400‑970, Brazil e-mail: [email protected] W. L. Araújo  Department of Microbiology, Institute of Biomedical Sciences, University of São Paulo, Av. Prof. Lineu Prestes, 1374 ‑ Ed. Biomédicas II, Cidade Universitária, São Paulo, SP 05508‑900, Brazil e-mail: [email protected] J. R. P. Parra  Department of Entomology and Acarology, Escola Superior de Agricultura “Luiz de Queiroz”, University of São Paulo, Av. Pádua Dias, 11, Piracicaba, SP 13418‑900, Brazil

impair larval development and decrease larval weight. Sugarcane seedlings were inoculated with 33.1:pJTT, and reisolation confirmed the capacity of 33.1:pJTT to continuously colonize the sugarcane. These results prove that P. agglomerans (33.1), a sugarcane growth promoter, can be improved by expressing the Cry protein, and the resulting strain is able to control the sugarcane borer. Keywords Endophyte · Pantoea agglomerans · cry gene · Sugarcane borer

Introduction Sugarcane is any of 6–37 species (depending on which taxonomy system is used) of tall perennial true grasses of the genus Saccharum, tribe Andropogoneae, native to the warm temperate to tropical regions of South Asia. Modern sugarcane cultivars are the products of successful crosses between species of the genus Saccharum that were made by breeders in the late nineteenth century (Matsuoka et al. 1999; Cheavegatti-Gianotto et al. 2011). Worldwide, sugarcane is one of the largely cultured crop. In 2012, FAO estimates it was cultivated on about 25.7 million hectares, in approximately 100 countries. Brazil was the largest producer of sugarcane in the world with a worldwide harvest of 6.71 million tons, followed by India, China and Thailand (FAOSTAT 2012). Economic interest in sugarcane has increased significantly in recent years due to the increased worldwide demand for sustainable energy production. It is estimated that the Brazilian production of sugarcane must double in the next decade to meet this goal. One important tool certainly is the biotechnological advances that might help to reduce the environmental impacts of increased sugarcane production, decreasing the injuries caused by pathogen and pests (Cheavegatti-Gianotto et al. 2011).

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The Gram-positive bacterium Bacillus thuringiensis (Bt) is currently used as a safe alternative to chemical pesticides, since it produces, during its sporulation, crystals with high specificity. These crystals are very toxic proteins known as δ-endotoxins, Cry toxins or Cry proteins. Although they have high toxicity, the Cry proteins are very selective due to the specificity of the receptor binding sites for the midgut epithelial cells of the target insect (Pigott and Ellar 2007). Among the many Cry protein family members, Cry 1 and Cry 2 are of particular interest in agriculture due to their high toxicity to insect larvae of the orders Lepidoptera and Coleoptera, and even nematodes and mites (Dean 1984; Wei et al. 2003; Kotze et al. 2005; de Macedo et al. 2012). Despite its success as a bio-insecticide, there are some problems with the application of Bt, such as difficulties in reaching the target larvae because many insect pests live inside the host plants (Downing et al. 2000). Therefore, a feasible alternative is the cloning of genes that code for Cry proteins into endophytic microorganisms living in the same niche as the insect pests (Fahey et al. 1991; Tomasino et al. 1995; Ragev et al. 1996; Barboza-Corona et al. 1999; Barboza-Corona et al. 2003; Choi et al. 2008). The sugarcane borer, Diatraea saccharalis (F.) (Lepidoptera: Pyralidae), is one of the most important sugarcane pests in Brazil, and it is widely distributed in sugarcane belts across the American continent (Dinardo-Miranda 2008). The larvae tunnel into the stalks, causing the death of a large number of young shoots and a sharp reduction in the productivity of more developed sugarcane crops. The infected stalks lose weight and become smaller and thinner, and many wither and die, while others are broken by the wind (Dinardo-Miranda et al. 2012). The presence of many microorganisms in sugarcane stalks is common, especially fungi that cause the “red rot” disease, which reduces the sucrose content in the stalks by converting it into glucose and fructose. The microorganisms present in the stalks contaminate the broth, thereby hampering industrial processes, hindering the attainment of high-quality sugar and inhibiting fermentation (Botelho and Macedo 2002; DinardoMiranda 2008; Parra et al. 2010). The Pantoea agglomerans endophytic strains are strong candidates for transformation with cry genes. This species has been well studied due to the potential benefits to agriculture as a control agent for pests and phytopathogens, as well as an important plant growth promoter (Liu et al. 1995; Ongena et al. 2000, Bonaterra et al. 2003; Jeun et al. 2004). Torres et al. (2013) suggested this species as a great candidate to deliver enzymes or other proteins in planta. Many reports have described endophytic colonization of sugarcane plants by this species (Nunez and Colmer 1968; Baldani et al. 1986; Cavalcante and Döbereiner 1988; Dong et al. 1994; Loiret et al. 2004). Recently, P. agglomerans 33.1, which was isolated from Eucalyptus

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grandis, was described as a sugarcane cross-colonizer able to promote sugarcane growth (Quecine et al. 2012). Therefore, we transferred cry1Ac7 to P. agglomerans (33.1) and demonstrated the improvement in its potential to increase sugarcane fitness by controlling the sugarcane borer.

Materials and methods Bacterial transformation, plasmids and culture conditions Pantoea agglomerans 33.1, a sugarcane cross-colonizer isolated from E. grandis plants (Procópio et al. 2009; Quecine et al. 2012), was transformed by electroporation with the plasmid pJTT, as described by Ferreira et al. (2008). 33.1 strain was grown (OD560 nm 1.0) for 1 day at 28  °C in 5 mL Luria–Bertani (LB) liquid medium (Sambrook and Russel 2001), harvested by centrifugation at 4  °C, resuspended with 1 mL of cold ultrapure water and electrophoresed (2.5 kV; 25 mA; 25 mF; and 400 Ω) with 0.1 mg of pJTT plasmid. The pJTT plasmid carries the cry1Ac7 gene, homology sites for bacterial chromosomal integration and a kanamycin resistance gene (Downing et al. 2000). The 33.1:pNKGFP strain (Ferreira et al. 2008) was used as the bacterial negative control in the bioassays against D. saccharalis due to its kanamycin resistance. All of the strains were stored in 20 % glycerol at −80 °C. Fresh cultures were started from glycerol stocks for each experiment by plating portions onto (LB) agar supplemented with the appropriate antibiotic and incubating at 28 °C for 24 h. Southern blot analysis was used to demonstrate the integration of the Omegon-Km–ptac-cry1Ac7 cassette into the chromosome of the P. agglomerans 33.1:pJTT clone used in the bioassays against D. saccharalis. Total bacterial DNA was extracted as previously described by Quecine et al. (2012). The probe was obtained according to Downing et al. (2000), and the hybridization was confirmed with the Gene Images™ AlkPhos Direct™ labeling and detection system (GE Healthcare) according to the manufacturer’s instructions. Insect rearing conditions All bioassays were conducted using D. saccharalis larvae that were routinely maintained on an artificial diet at the Laboratory of Insect Biology of the Department of Entomology and Acarology, University of São Paulo, Piracicaba, SP, Brazil. The insects were fed with an artificial diet (King and Hartley 1985) according to Parra (1996) and maintained in an environmentally controlled room under the following conditions: 25 °C, 60 % relative humidity and 14-h photophase. All in vitro bioassays were conducted under the same artificial diet and rearing conditions.

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In vitro 33.1:pJTT toxicity bioassay The 33.1:pNKGFP and 33.1:pJTT strains were inoculated into LB medium (50 ml), supplemented with kanamycin (50 μg/ml) and incubated at 28 °C with shaking (150 rpm) until the late log phase. The cells were harvested (at 4,500g for 15 min) and washed with phosphate-buffered saline [PBS, containing (g/L) Na2HPO4 1.44; KH2PO4 0.24; KCl 0.20; NaCl 8.00; pH 6.5]. The cooked insect artificial diet was chilled to 55 °C, and 107 CFU/ml of each strain was added to it. The culture medium without bacterial cells was used as a control. The diet was poured into petri dishes. After 24 h, 10 D. saccharalis larvae (second instar) were added per plate. The larvae grew in these plates for seven days after the inoculation (DAI), and then, the larval mortality was evaluated. Two independent experiments were performed using six replicates for each experiment. The cooked artificial diet (10 ml) supplemented with kanamycin (50 μg/ml) and the 33.1:pJTT strain, which was added in two different concentrations (102 and 107 CFU/ ml), were also poured into glass tubes covered with cotton to allow air exchange. Two larvae (second instar) were transferred per tube, and 20 tubes were used per treatment. The larvae were allowed to grow to the pupal stage. Several parameters were measured: (1) larval mortality, (2) the duration of larval development and (3) pupal weight. These parameters were evaluated every two days until all surviving larvae reached the pupal stage. The density of the 33.1:pJTT bacteria was measured in the artificial diet and larvae by re-isolation 24 h after inoculation. In vivo bioassay For the in vivo bioassay, we used the sugarcane variety SP80-1842 cultivated in the experimental field of Department of Genetic, ESALQ-USP, Piracicaba, SP. The plants were supplied by Dr. Sabrina Moutinho Chabregas (CTC, Piracicaba, SP, Brazil). The stalks from 1-year-old sugarcane were cut (approximately 2.5 cm). The 33.1 wild-type, 33.1:pNKGFP and 33.1:pJTT strains were inoculated into 500 ml of LB medium supplemented with kanamycin (50 μg/ml) (except the wild type, for which kanamycin was not used) and incubated at 28 °C with shaking (150 rpm) until the late log phase. The autoclaved stalks (121 °C for 15 min) were then immersed in 500 ml of bacterial culture (107 CFU/ml) supplemented with the following anti-contaminants: kanamycin (50 μg/ml), except for the wild type; benomyl benzimidazole (50 μg/ml); and ascorbic acid (0.1 %) to avoid oxidation. The stalks were incubated for 1 h at 28 °C under rotation at 100 rpm and then transferred to a glass container, which had been previously sterilized. After 24 h at 28 °C to allow the establishment of the bacteria, 15 larvae (second instar) were transferred to each stalk

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and incubated at 28 °C with a 14-h photophase. Although the possible autoclaving affects the structure of the sugarcane stalks, the larvae fed the plant material. The culture medium without bacterial cells was used as a control. The following parameters were measured at 10 and 30 DAI: (1) mortality level, (2) larval weight and (3) bacterial density in larvae and stalks. The experiments were performed using five replicates. Sugarcane colonization by 33.1:pJTT The micropropagated sugarcane seedlings of variety SP801842 were transferred to plastic pots containing the organic substrate Eucatex PlantMax® Horticultura (Eucatex). First, the transferred plants were acclimated in a humid chamber at 28 °C for seven days. The 33.1:pJTT cells in log growth phase were inoculated into the substrate (108 CFU/ml), and the pots were transferred to a greenhouse at 28 °C. As a control, the substrate was inoculated with PBS without bacterial cells. Five plants for each treatment were sampled to determine the density of the 33.1:pJTT bacteria in the rhizosphere and inside the sugarcane root and aerial part tissues at 30 DAI. Bacterial re‑isolation For bacterial re-isolation, samples of larvae (in vitro assay) and stalks (in vivo assay), as well as sugarcane tissues (greenhouse assay), were washed in running tap water and surface-disinfected [70 % ethanol for 1 min, sodium hypochlorite solution (2 % available Cl_) for 2 min, 70 % ethanol for 1 min and two washes in sterilized distilled water] according to the method of Araújo et al. (2001). To confirm the efficiency of the disinfection process, aliquots of the sterile distilled water used in the last washing were spread onto 5 % tryptic soy agar medium and examined for surface contaminants after three days of incubation at 28 °C. The surface-disinfected samples, as well as the artificial diet portion and rhizosphere (1 g), were macerated in sterile PBS and maintained at 28  °C under 150 rpm agitation. Appropriate dilutions were subsequently plated onto 5 % TSA supplemented with kanamycin (50 μg/ml) and benomyl (50 μg/ml) fungicide to prevent fungal growth. The number of 33.1:pJTT cells was calculated as CFU/g of sample after two days of incubation at 28 °C. Statistical analysis Data analysis was carried out with the SAS software package (SAS Institute Inc., Cary, NC, USA). A completely random design was used for all assays. The effects of the 33.1:pJTT on sugarcane borer larval mortality were determined by counting the number of insect larvae. Statistical analysis was

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applied to the total number of surviving larvae. An ANOVA of the regression curve for the number of larvae that reached the pupal stage was obtained by a stepwise regression that was used to determine which varieties provided the best fit. The re-isolation bacterial count data were transformed using log10 of x + 2 before implementing an ANOVA.

the plasmid pJTT. Among the obtained transformants, one clone (33.1:pJTT) was selected randomly for Southern blot analysis and further toxicity bioassays against D. saccharalis larvae. The molecular analysis confirmed the integration of the Omegon-Km–ptac-cry1Ac7 cassette into the chromosome of 33.1:pJTT. The wild type did not show the Km–ptac-cry1Ac7 fragment using the same probe (Fig. 1).

Results

In vitro bioassay

Pantoea agglomerans 33.1 transformation with pJTT

Fig. 1  Southern blot analysis of plasmid pJTT, 33.1 and 33.1:pJTT chromosomal DNA cut with EcoRI and probed with the 4-kb BamHI fragment of pJTT carrying the ptac-cry1Ac7 cassette. The lanes correspond to the following bacteria: lane 1 P. agglomerans 33.1 wild type; lane 2 P. agglomerans 33.1:pJTT; and lane 3 plasmid pJTT

The mortality of D. saccharalis larvae fed with the artificial diet supplemented with 33.1:pJTT (107 CFU/ml) was significantly higher than those fed with the control or 33.1:pNKGFP supplement at the same concentration (Fig. 2a). This mortality was dependent on the bacterial density, since at a high dose (107 CFU/ml), the larval development was affected until the pupal stage, while at a low dose (102 CFU ml), no effect was observed, and the mortality was statistically similar to that of the control larvae (Fig. 2b). Under the gnotobiotic condition, 33.1:pJTT had an effect on the D. saccharalis larvae, increasing the number of dead larvae as follows: control 0 %; low dose 11.1 %; and high dose 27.3 % of larval mortality. However, we did not observe differences in pupal weight means based on the treatment: control (0.158 ± 0.053); low dose (0.149 ± 0.055); and high dose (0.165 ± 0.045). After re-isolation, the 33.1:pJTT cells were detected in the larvae (1.000 CFU/larva), proving that the bacteria were ingested by the larvae fed with an artificial diet supplemented with bacterial cells. The maceration and dilution of this diet in PBS was very difficult due to material consistency. The bacterial cells probably keep aggregated in the diet, hampering the

Fig. 2  Effects of P. agglomerans 33.1:pJTT on D. saccharalis larvae during in vitro bioassays. a The larval mortality was measured 7 DAI of D. saccharalis larvae fed with an artificial diet supplemented with 107 CFU/ml of the 33.1:pNKGFP or 33.1:pJTT strains. The mortality is represented as the mean percentage of dead larvae for six replicates. Each replicate contained 10 larvae. Values with the same letter are not significantly (P > 0.05) different according to Tukey’s test. b Larval development of D. saccharalis from the first instar until the pupal stage. A high dose (107 CFU/ml) and low dose (102 CFU/ml) of

33.1:pJTT were added to the artificial diet of the insects. The percentage of larvae that reached the pupal stage was determined from the total number of surviving insects. The statistical difference between the three curves and the regress equation was obtained by ANOVA. The result of the diet with high-dose (filled triangle) equation curve y  = 346.55ln(x)–1107.7 (R2  = 0.9227) is statistically different (P > 0.05) from both the diet with low-dose (filled square) equation curve y  = 306.33ln(x)–957.04 (R2  = 0.9378) and the control (filled diamond) equation curve y = 328.74ln(x)–1025.2 (R2 = 0.9311)

Pantoea agglomerans, strain 33.1, was transformed with a low efficiency (4.102 transformants/μg of DNA) using

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Fig. 3  Effects of P. agglomerans 33.1 strains on D. saccharalis larvae fed with sugarcane stalks. a aThe larval mortality was measured 10 and 30 DAI of D. saccharalis larvae fed with sugarcane stalks inoculated with 33.1, 33.1:pNKGFP and 33.1:1:pJTT. The results are expressed as the means of five replicates (15 larvae per stalk) for each treatment. bThe values are the means of 20 larval weights per treatment, measured 10 and 30 DAI. cThe bacterial density measured by re-isolation 10 and 30 DAI. The abundance data, in CFU/larva or

CFU/g of stalks, were log-transformed to log (CFU + 2) to normalize the variance. The results are expressed as the means of five replicates for each sample. dThe replicates were composed of 5 larvae each. nd not determined. For all data, the values annotated with the same letter within a column are not significantly (P > 0.05) different according to Tukey’s test. b Larvae fed with 33.1:pJTT-inoculated sugarcane stalks (above) and larvae fed with the control non-inoculated stalks (below) c Dead larva from a 33.1:pJTT-inoculated sugarcane stalk

re-isolation; thus, we were not able to re-isolate 33.1:pJTT cells from the diet, independent of the applied dosage.

No differences were observed in the number of 33.1:pNKGFP and 33.1:pJTT cells re-isolated at either evaluated time in stalks. However, the number of re-isolated bacteria 33.1:pJTT from larvae 10 DAI was very low compared with 33.1:pNKGFP. This fact may be the effect of very well-related septicemia due to the Cry proteins expressed by the bacterial cells.

In vivo bioassay After the in vitro 33.1:pJTT toxicity bioassays, an in vivo bioassay was performed to simulate the real application of 33.1:pJTT as a possible control measure for D. saccharalis larvae in the field. The mortality of the sugarcane borer fed with 33.1:pJTT-inoculated stalks was statistically higher than in the other treatments at all of the evaluated times. The larval mortality at 30 DAI was higher in all treatments than that at 10 DAI, most likely due to stalk desiccation (Fig. 3a). We also observed that 33.1:pJTT had a negative effect on larval weight (Fig. 3b). During the evaluation, we observed that many dead larvae that were fed with 33.1:pJTT stalks were completely deteriorated and of a dark color, which is a typical characteristic of Cry protein exposure (Fig. 3c). These results prove the consistency of the developed methodology. Interestingly, the larval cycle was longer for the larvae feeding on the stalks than those on the artificial diet. At 30 DAI, no insects had reached the pupal stage, while at 32 DAI in vitro, all of the surviving larvae had reached the pupal stage. Differences were observed in the number of bacteria reisolated from the artificial diet and the sugarcane stalks.

Sugarcane colonization by P. agglomerans 33.1:pJTT The ability of 33.1:pJTT to colonize sugarcane was confirmed by re-isolation of the bacteria 30 DAI. The bacterial density in the sugarcane rhizosphere, root and aerial parts was measured. We observed a higher density of 33.1:pJTT in the rhizosphere than the root and aerial parts, demonstrating that the introduction of an exogenous gene did not affect the sugarcane–33.1 interaction (Fig. 4). The 33.1:pJTT strain retained the capacity to efficiently survive in the rhizosphere and colonize all of the sugarcane tissues.

Discussion Currently, the improvement in sugarcane production is a challenge; many biotic factors, such as the presence of

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Fig. 4  P. agglomerans 33.1:pJTT density during sugarcane colonization, measured by re-isolation 30 DAI. The abundance data, in CFU/g of sample, were log-transformed to normalize the variance. The results are expressed as the means of four replicates for each sample. The values annotated with the same letter did not differ statistically (P > 0.05) according to Tukey’s test

pests, contribute to economic losses. The application of natural enemies, such as Cotesia flavipes, to control D. saccharalis has been an efficient alternative to the use of chemicals (Botelho and Macedo 2002; Parra et al. 2010, Veiga et al. 2013). However, residual injuries to 10 % of the crop justify the adoption of other strategies to control this pest, such as the use of the δ-endotoxins from Bt, using the principle of integrated management of pests (Arencibia et al. 1997; Falco and Silva-Filho 2003; Parra et al. 2010). Zhang et al. (2013) demonstrated that Diatraea saccharalis has different levels of susceptibility to Bt proteins, Cry1Ab, Cry1Aa, Cry1Ac and Cry1F, suggesting that pyramiding these two types of Cry proteins into a plant could be a good strategy for managing D. saccharalis. The cloning of diverse cry genes has allowed for the extensive exploration of the biotechnological potential of the Cry proteins because novel Cry proteins can be used to develop new recombinant bacterial strains (Xue et al. 2008). The application of transgenic microorganisms harboring the cry gene as biocontrol agents has some advantages for plants: the fast obtaining of recombinants and synthetic codons are not needed because the bacterial genomes are more similar to the Bt genome than to the plant genome, and transgenic plants expressing the cry genes have been created (Perlak et al. 1991; Estruch et al. 1997; Fearing et al. 1997). However, the only commercial product based on a genetically modified bacterium expressing a cry gene is “Incide,” which contains the bacterium Clavibacter xyli subsp. cynodontis to control the corn borer, Ostrinia nubilalis (Fahey et al. 1991; Tomasino et al. 1995), demonstrating the huge demand for commercial products of this kind. Consequently, the cry genes have been introduced into many other bacteria species: Escherichia coli (Ge et al.

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1990), C. xyli (Turner et al. 1991), P. fluorescens (Downing et al. 2000), Burkholderia cepacia, Bacillus megaterium (Bora et al. 1994), B. cereus (Moar et al. 1994), Azospirillum lipoferum and A. brasilense (Udayasurian et al. 1995); Gluconacetobacter diazotrophicus (Falcão-Salles et al. 2000), H. seropedicae (Downing et al. 2000, Falcão-Salles et al. 2000), B. subtilis and B. licheniformis (Theoduloz et al. 2003); Methylobacterium extorquens (Choi et al. 2008); and others. However, this is the first report of the manipulation of a strain of P. agglomerans by the addition of a cry gene to control an insect. Pantoea agglomerans (33.1) has been described as an indole acetic acid producer, as well as a phosphate solubilizer, which accounts for the increase (by more than 30 %) in the dry mass of the sugarcane aerial parts after inoculation with this bacterium. The monitoring of this strain also proved that it is able to colonize sugarcane tissues, including the aerial parts—the same niche as the sugarcane borer (Quecine et al. 2012). Therefore, to further the benefit of 33.1 to sugarcane fitness, we used the plasmid pJTT, which was developed by Downing et al. (2000). These same authors introduced the cry1Ac7 gene into the phylospheric bacterium P. fluorescens (14) and the diazotrophic endophyte Herbaspirillum seropedicae (HRC54) to control Eldana saccharina, a sugarcane borer in South Africa just in vitro conditions. The 33.1:pJTT had the cry1Ac gene integrated in its genome; the stability of integration was observed after 180 generation (data not shown), being it important to further field assays. The cry1ac7 expression by the 33.1:pJTT was confirmed indirectly. The In vitro bioassay 33.1:pJTT had a significant negative effect on the larval surviving and their development. Similar results were obtained using 33.1:pJTT-inoculated sugarcane stalks to feed the D. saccharalis larvae. The present work also shows a new methodology to test the effects of this bacterium to control sugarcane borer simulating the crop condition, inner sugarcane stalks. Our assay of equivalence confirmed that the cassette OmegonKm–cry integrated in 33.1:pJTT genome did not affect the bacterial capacity to colonize sugarcane. These results clearly give new perspectives to a possible control of sugarcane borer inside of the plant by the heterologous expression of cry1ac7 by 33.1:pJTT as proved in vivo bioassay. However, a detailed study of its impacts on the environment should be researched as well new assays, confirming the capacity of 33.1:pJTT to promote sugarcane growth as the 33.1 wild type to confirm the safety and the advantages of a further application of this strain in sugarcane fields. Acknowledgments  This work was supported by grants from the São Paulo Research Foundation (FAPESP) (Proc. No. 05-53748-6 and 02/14143-2) and The National Council for Scientific and Technological Development (CNPq). We also thank Neide Graciano Zério for collaborating on the in vitro bioassays and Dr. Sabrina Moutinho

Arch Microbiol (2014) 196:227–234 Chabregas from CTC (Piracicaba, SP, Brazil) for discussions and for supplying the sugarcane plants.

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