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Caenorhabditis elegans as an indicator of toxicity of Bacillus thuringiensis strains to Meloidogyne incognita race 3/Caenorhabditis elegans como indicador de toxicidade de estirpes de Bacillus thuringiensis a Meloidogyne incognita raca 3.

INTRODUCTION

Cotton (Gossypium hirsutum L.) is grown in more than 60 countries, covering an area of more than 35 million hectares and producing around 25 million tons of plume per year, and is; therefore, one of the most important agricultural commodities in the world (ABRAPA, 2015).

Soil and climatic conditions of the producing regions, associated with their cultivation systems, sometimes in extensive areas and with few cultivated varieties, facilitate the appearance of diseases (SUASSUNA & COUTINHO, 2007; AMORIM et al., 2011). Among them, root-knot nematode caused by Meloidogyne incognita race 3 stands out. This nematode, after penetrating the host root, moves to the root cortex where it establishes the infection site, initiating the feeding process (MOENS et al., 2009). It remains at this site feeding and undergoing several ecdysis until emerging as a male or female adult (EISENBACK & TRIANTAPHYLLOU, 1991; MOENS et al., 2009). This characteristic offers several benefits, such as protection against inclement weather and natural enemies, and makes it difficult to develop effective control methods. The rich food available favors its reproduction and a single female can produce from 200 to 1,000 eggs (FREIRE, 2007). M. incognita stands out today as the main plant-parasitic nematode of the cotton crop in Brazil, causing losses of more than 40% in highly infested areas (FREIRE et al., 2015).

Among the control methods are the prevention of pathogen entry into areas free of infection, use of resistant varieties, seed nematicide applications, solarization, deep plowing, crop rotation and incorporation of organic residues and the use of antagonists (FERRAZ et al., 2012). There is a wide range of rhizosphere bacteria antagonistic to phytopathogens that colonize roots of plants, and act as bio-controllers producing substances toxic to pathogens (LUZ, 1996;TURAet al.,2007; MACHADO et al., 2012). However, no method is fully effective.

Bacillus are recognized as bioregulators and natural antagonists of several phytopathogens, in addition to promoting growth and inducing resistance in plants (MAHDY et al., 2001; VONDERWELL et al., 2001; ONGENA & JACQUES, 2007; RUSSI, 2012). These bacteria are gram-positive, spore forming, and some have the ability to produce protein crystals during their sporulation phase, such as B. thuringiensis. Proteins contained in these crystals may be toxic to insects of different orders and to nematodes, as is the case of the previously described nematocidal proteins Cry5B, Cry6A, Cry 14A, Cry21A and Cry55A (WEI et al., 2003; MONNERAT et al. 2001; GUO et al., 2008). There is great demand to identify and select bacterial strains with high control activity on M. incognita to enable their use as tools in biological control programs. However, in vitro and in vivo assays with this nematode are hampered by the difficulty in obtaining large populations in a short period of time.

In addition, in vivo tests should be performed to verify results obtained in vitro and are quite laborious and time consuming, as the cycle of M. incognita lasts about 28 days and to be safe in the evaluations of the experiments, they should be conducted for approximately 90 days. In addition, M. incognita is an obligate plant parasite, making it difficult to use in in vitro selection trials. Thus, it would make sense to substitute this nematode with an organism that can be more easily managed. Caenorhabditis elegans, a free-living nematode inhabitant of moist soils that uses atmospheric oxygen and feeds on bacteria, appears to be a plausible alternative (BRENNER, 1974). This nematode is now used as a model organism for the most varied types of research (SCHIERENBERG & WOOD, 1985; DONALD, 1997). The present research aimed to evaluate the potential of C. elegans as an indicator organism for selection of strains of B. thuringiensis with biocontrol potential against M. incognita Race 3.

MATERIALS AND METHODS

Bacillus thuringiensis strains

Bacterial strains used in these experiments were isolated from soil from different regions of Brazil using the methodology of World Health Organization, (1985), they were: S906, S1185, S1192, S2036, S2038, S2193, S2493 and S2496 (all strains are deposited in the Invertebrate Bacteria Collection of Embrapa Genetic Resources and Biotechnology). These bacteria were cultured in Erlenmeyer flasks (500mL) containing 150mL of Embrapa medium (MONNERAT et al., 2007), in a rotary incubator at 150RPM (28[+ or -] 2[degrees]C) for 72h. After this culture period the morphology of strains was analyzed with a phase contrast microscope to observe the presence of the protein crystal (MONNERAT et al., 2001) and its concentration was quantified according to the methodology of ROMEIRO (1989) and then adjusted with saline solution to 3X[10.sup.7][mL.sup.-1].

Maintenance of the C. elegans population

Maintenance of the C. elegans colony was carried out according to the protocol of STIERNAGLE (2006) with modifications. This nematode was given to Embrapa by Dr. Colin Berry of Cardiff University. The nematode feeding was done using E. coli bacteria, OP50 strain, multiplied in a rotary shaker (150RPM, 36[degrees]C, 12h) in Luria-Bertani medium (LB) (SAMBROOK & RUSSELL, 2001). Every two days 1mL of the bacterial suspension was given to the nematodes in the culture plates. The colonies of C. elegans were kept in an incubator (Marconi, mod. MA 403) in the absence of light (21[degrees]C).

Maintenance of the population of Meloidogyne incognita

The initial inoculum of M. incognita Race 3, provided by the Phytonematode Laboratory at Embrapa Genetic Resources and Biotechnology, were multiplied in tomato plants (Solanum lycopersicum cv. Santa Clara) for 3 months in greenhouse. After this period, eggs were extracted from the plants according to the method described by HUSSEY & BARKER (1973), modified by BONETI & FERRAZ, (1981). After extraction, eggs were surface disinfected by a method described by ZUCKERMAN & BRZESKI (1966). Eggs were suspended in 30mL of commercial PerioGard[R] product (chlorhexidine gluconate solution 0.12%) with antibiotics added (10 [micro]g/mL erythromycin, 2.5g/L streptomycin) for 30min. They were then centrifuged (3min, 360g), and after this, the supernatant was discarded and the eggs present in the pellet were suspended in sterile distilled water. The procedure was repeated twice. In a sterile environment eggs were placed in a modified Baermann funnel (FLEGG, 1967) for hatching. Second-stage juveniles (J2) of M. incognita were collected every 2 days in a sterile environment and placed in a capped vial under refrigeration (4[+ or -]2[degrees]C).

Selection of strains toxic to C. elegans

The assay was performed in Petri dishes (90X15mm) to which 7.5mL of the C. elegans suspension (concentration of 300 nematodes/mL) and 2.5mL of the bacterial suspension (concentration of 3X[10.sup.7]CFU/mL) were added. As controls, pure Embrapa medium (EM), Embrapa medium (MONNERAT et al., 2007) plus erythromycin (10 [micro]g/mL), carbofuran (a liter of the comercial product/20L of water) and saline solution (8,5g of HCl/water liter) were used. Plates were identified, dated and their edges were sealed with plastic film, to avoid contamination. The assay was incubated in the dark at 21[degrees]C for 48h. After this period, number of nematodes was evaluated with an optical microscope. For this, 2mL of the contents of each plate was collected, diluted 30 times (in tap water) and with the help of a Peters chamber and an optical microscope, counts of live individuals (they are very agile and easily identified under an optical microscope) were performed in the suspension. For each plate three counts were performed, and the mean of these counts was used for statistical analysis.

The experiment was a completely randomized in design with three replicates per treatment. The data obtained from the number of live nematodes per plates were transformed into [square root]x, to facilitate the statistical analyses, submitted to analysis of variance and the means compared by Duncan test (P[less than or equal to]0.05) [Assistat version 7.7 (SILVA & AZEVEDO, 2014)].

This bioassay was performed using nematodes obtained with the Baermann funnel, following the same methodology as described for C. elegans.

Selection of strains toxic to M. incognita Race 3 in vivo

This experiment was carried out under greenhouse conditions. Tomato seedlings (Solanum lycopersicum cv. Santa Clara) produced in Styrofoam trays were transplanted one week after emergence into 2-liter vessels with Bio-Plant[R] compost (sphagnum peat, coconut fiber, rice husk, pine-bark, vermiculite, and nutrients) and autoclaved soil (1:1). One week after transplantation, the seedlings were inoculated, with the aid of micropipette, with 5mL of the bacterial suspension (3x[10.sup.7]CFU/mL), this suspension was inoculated in the soil next to the base of seedlings. For the controls, distilled water was inoculated instead of suspension of bacteria. Five days after the first inoculation, nematode inoculation was performed. For this, a suspension containing 10,000eggs was deposited in three equidistant furrows dug approximately 2cm deep. Immediately after inoculation of the nematodes, the bacteria were again inoculated as at the start of the assay. Plants were kept in a greenhouse for a period of three months (28[degrees]C [+ or -] 80% RH). Irrigation was performed daily at the end of the afternoon and monthly fertilizations used three grams of the Dimy[R] (granulated commercial fertilizer 04-14-08). Weekly thinning of senescent leaves was performed to avoid pest emergence; flowers were also removed to avoid fruit production.

The assay was evaluated after 3 months. The aerial part of the plants was discarded and the root system were taken to the Laboratory of Nematology where eggs were extracted according to the method of HUSSEY & BARKER (1973) modified by BONETI & FERRAZ, 1981). Egg suspensions were homogenized and with a Pasteur pipette, 2mL of the suspension were transferred to beakers (100mL) and the volume was adjusted to 60mL with water. Three samples of 1mL of each suspension were counted in a Peters chamber and the mean of these was used to calculate egg concentration and reproduction factor (OOSTENBRINK, 1966).

The experiment was a completely randomized in design with four replicates per treatment. The data obtained from the RF calculation were transformed into [square root]x, to facilitate the statistical analyses, submitted to analysis of variance and the means compared by Duncan test (P[less than or equal to]0.05) [Assistat version 7.7 (SILVA & AZEVEDO, 2014)]. All the assays were replicate two times.

Molecular characterization of toxic strains

The strains were characterized for the presence of cry genes of B. thuringiensis. The procedure of DNA extraction of the selected bacteria to carry out the Polymerase Chain Reaction was done as described by BRAVO et al. (1998), in which the bacteria were cultured in solid Embrapa medium (MONNERAT et al., 2007) for 16h at 30[degrees]C. After this period, for each sample, the bacterial growth was collected with a loop and transferred to a 1.5mL polyethylene tube properly identified and containing 200[micro]L of sterile ultra-purified water (Mili Q). Samples were then homogenized in Vortex apparatus and frozen at -80[degrees]C for 20min and then boiled in a 100[degrees]C water bath for 10min and finally incubated on ice for 2min. The supernatant obtained was used for reactions. Different oligonucleotides were used in the PCR reaction, 5[micro]L of the DNA from each strain was transferred to a 0.2mL polyethylene tube containing 0.5[micro]M of each oligonucleotide, 0.2mM dNTP, 1X Taq buffer and 2.5U of Taq DNA polymerase (5.0U), totaling a final volume of 30[micro]L. Oligonucleotides were used to identify the following genes: cry1, cry2, cry3, cry4, cry5, cry8, cry9, cry 10, cry 11, cry 12, cry13, cry14, cry17, cry19, cry21, cry24, cry25, cry32, cry39, cry40, cyt1 and cyt2. PCR conditions were performed for each oligonucleotide as described by CERON et al. (1994), CERON et al. (1995), BRAVO et al. (1998) and IBARRA et al. (2003). For analysis of the result, 25[micro]L of the mixed PCR product was applied to 5[micro]L of 10X run buffer in 1.5% agarose gel. Electrophoresis run was done in TBE 1X buffer (Tris base; boric acid; 0.5M EDTA-pH 8.0). Subsequently, the gel was stained with ethidium bromide at 1[micro]g/mL for 20min and followed by distilled water for 15min. The gel was visualized by transilluminator under UV light and photo-documented (Eagle Eye, Stratagene).

RESULTS AND DISCUSSION

Assay with C. elegans in vitro and M. incognita in vitro and in vivo

Of the eight B. thuringiensis strains used in this assay, six were toxic to C. elegans. The other two matched the control. The highest response was presented by strain S906 with greater effectiveness than the control (saline solution), and reduction by more than 51% of the nematode population, followed by strain S2493 with 41.8% control. Other strains caused a reduction in the nematode population ranging from 14.7 to 25.6% of the control.

Molecular characterization of strains showed that 4 of them had known cry genes and 4 of them did not present expected PCR products for the detection of cry genes. As all showed protein crystals when observed in phase contrast microscopy, it is likely that other genes are involved in the formation of the protein evidenced by the presence of crystals. The most toxic strain S906 was one of the strains that despite presenting crystals, did not present expected PCR products with the primers tested. In addition, the S2493 strain, which resulted in a reduction of 41.8% in the nematode population, shows the cry6 gene, which encodes the Cry6 toxin described as a nematicide (BRAVO et al., 2012; PALMA et al., 2014), corroborating the results we obtained in this assay. SILVERA & LENGUA (2015) tested the activity of Cry6 on gastrointestinal nematodes of sheep (Nematodirus spathiger), reporting control of 44.7% and 45.6% in eggs and larvae, respectively. The strain S2038 presented the cry11 gene, which encodes proteins that are toxic to mosquito larvae (FERNANDEZ et al., 2005), and recently reported as toxic to Haemonchus contortus (LARAet.al., 2016). Strains S2193 and S2496 did not differ from the control, presenting cry1, cry2 and cry52 genes that encode proteins that were not described as toxic to nematodes. Other strains that had an antagonistic effect on the nematode (S1192, S1185 and S2036) did not show PCR products for the genes tested, indicating that these strains should have toxin genes different from those known. The nematicide carbofuran presented a statistically superior result to the other treatments.

The in vitro test results with M. incognita (Table 1) showed the strains caused mortality, between 4 and 41.1%, lower than that obtained in the C. elegans assays. The S2036, S1192 and S906 strains caused mortality higher than the control, respectively of 41.1, 19.7 and 24.9%. However, because the protein crystal is too large to be ingested by M. incognita, making it impossible for it to be toxic, it is possible that the toxicity reported is attributed to other substances produced, such as enzymes, which may be acting through contact with the nematode (URWIN et al., 1997). In the in vivo assay, four strains (S2036, S1185, S1192 and S906) reduced the nematode population from 17.81 to 46.79%. This fact was also evidenced by the reproduction factor (RF), which ranged from 9.74 to 10.15 (Table 1).

The three strains toxic to M. incognita in vitro were toxic to M. incognita in vivo and C. elegans. Results obtained in the bioassay with M. incognita in vivo were more similar to those obtained with C. elegans than with M. incognita in vitro. This can be explained by the fact that M. incognita in vitro does not feed on the Cry toxins of B. thuringiensis. In contrast, studies showed that strains of B. thuringiensis can colonize plants in a systemic way and facilitate the acquisition of toxin by pests (MONNERAT et al., 2009). Complementarily, C. elegans, being free-living and feeding on bacteria, and having a greater movement on the plates, has more contact with the bacteria and proteins dispersed in the medium in which it is found. Devidas & Rehberger (1992) working with M. incognita and C. elegans in vitro also noticed a greater sensitivity of the free-living nematode to the compounds produced by B. thuringiensis and attributed this fact to C. elegans' dietary habits, which predispose it to another mode of action of the Bt toxins where they would act on its gut and not just on the epidermis.

In all three assays the M. incognita toxic strains were also toxic to C. elegans and that those that were not toxic to M. incognita were also not toxic to C. elegans. Some toxic to C. elegans were not toxic to M. incognita. Conversely, no strain that killedM. incognita both in vivo and in vitro killed C. elegans.

Results allow us to infer that C. elegans can be an indicator of toxicity of B. thuringiensis strains to nematodes and this methodology can be useful when it is desired to carry out a selection of strains from B. thuringiensis collections.

CONCLUSION

The M. incognita toxic strains (S906, S1192, S2036) of B. thuringiensis were toxic to C. elegans and those that were not toxic to M. incognita were not toxic to C. elegans. In addition, C. elegans can be an indicator of toxicity of B. thuringiensis strains to M. incognita and this approach can be useful when it is desired to carry out a selection of strains from B. thuringiensis collections.

http://dx.doi.org/10.1590/0103-8478cr20170712

Received 10.06.17 Approved 05.04.18 Returned by the author 06.12.18

ACKNOWLEDGMENTS

The authors thank the Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq) and to the Coordination for the Improvement of Higher Education Personnel (CAPES).

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Sandro Coelho Linhares Montalvao (1) Marcelo Tavares de Castro (2) Carlos Marcelo Silveira Soares (3) Luiz Eduardo Bassay Blum (1) Rose Gomes Monnerat (4) *

(1) Departamento de Fitopatologia, Universidade de Brasilia (UnB), Brasilia, DF, Brasil.

(2) Faculdade de Agronomia e Medicina Veterinaria, Universidade de Brasilia (UnB), Brasilia, DF, Brasil.

(3) Instituto Mato-grossense do Algodao (IMA-Mt), Primavera do Leste, Mato Grosso, Brasil.

(4) Laboratorio de Bacterias Entomopatogenicas, Embrapa Recursos Geneticos e Biotecnologia, 72 Brasilia, DF, Brasil. E- mail: rose.monnerat@embrapa.br.

* Corresponding author.
Table 1--Number [transformed ([square root of x])] of Caenorhabditis
elegans in vitro and Meloydogine incognita in vitro and in vivo (NN),
percentage of control (%C) and genes present in Bacillus thuringiensis
strains.

Treatments       cry gene   Presence         C. elegans
                            protein
                             crystal        NN        % C

Control EM +
erythromycin        --                    81.9 a      --
Control EM          --                   78.3 ab      --
Initial             --                   71.4 cd      --
inoculum
Control saline      --                  76.1 bc *    0,0 *
solution
S2496             cry52         +        72.4 cd      9.5
                 cry1Aa,
                 cry1Ab,
                 cry1Ad,
S2193             cry1C,        +        72.1 cd     10.2
                  cry1D,
                  cry1F,
                  cry2Ab
S907                --          +        70.6 cd     13.6
S2036               --          +        70.2 de     14.7
S1185               --          +        68.1 de     19.5
S1192               --          +        67.3 de     21.6
S2038             cry11         +         65,6 e     25.6
S2493              cry6         +         58.0 f     41.8
S906                --          +         53.2 f     51.1
Carbofuran          --                    42.7 g     68.7

Treatments       M. incognita           M. incognita
                   in vitro               in vivo
                      NN         % C         RF          % C

Control EM +
erythromycin       13.7 ab       --      12.33 a *      0.0 *
Control EM          14.3 a       --          --          --
Initial                          --          --          --
inoculum
Control saline    13.5 ab *     0.0 *        --          --
solution
S2496              12.8 ab      10.6      10.84 ab      22.69
S2193              13.1 ab       6.7      10.32 ab      30.02
S907               12.8 ab       9.7       8.99 d       46.79
S2036               10.4 e      41.1      10.15 bc      32.21
S1185              13.3 ab       4.0      9.32 cd       44.82
S1192              12.1 cd      19.7      9.89 bc       35.64
S2038              14.0 ab       0.0      11.14 ab      17.81
S2493              12.6 ab      13.8      10.88 ab      22.10
S906               11.7 de      24.9      9.74 bc       37.64
Carbofuran          0.0 f        100         Nr          Nr

Values followed by the same letter do not differ by Duncan's test
(P[less than or equal to]0.05). The original data was transformed
([square root of x). * Reference value.
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Title Annotation:CROP PROTECTION
Author:Montalvao, Sandro Coelho Linhares; de Castro, Marcelo Tavares; Soares, Carlos Marcelo Silveira; Blum
Publication:Ciencia Rural
Date:Jul 1, 2018
Words:4453
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