Effectiveness and persistence of biological control of nematodes in sugarcane

The objective this study was to investigate the effect of use of Bacillus subtilis to control nematodes, considering the hypothesis that the efficacy and persistence of biological control is equivalent to chemical control with respect to the community of Meloidogyne spp. and Pratylenchus zeae in soil during the growth of sugarcane (Saccharum spp.). For this study, soil was collected from cultivated area of sugarcane with a history of nematode infestation. The following treatments were established: control, B. subtilis (AP-3), B. subtilis (PRBS-1) and chemical nematicide (carbofuran). Evaluations of nematodes in soil were taken in intervals of 15, 30, 45, 60 and 75 days, respectively after planting. The application of B. subtilis in the soil caused the reduction of nematode reproduction factor evaluated in the soil, but also reduced the presence of nematodes in the final evaluation in the roots of sugarcane, similar to chemical treatment (carbofuran). In this study, it was concluded that biological treatment was equivalent to the chemical treatment on the efficacy of nematode control on sugarcane. The persistence of suppressiveness in Meloidogyne spp. and P. zeae provided by the treatments with B. subtilis and carbofuran was confirmed during the study.


INTRODUCTION
Sugarcane is a very important culture of both the socioeconomic point of view and from an environmental point of view.Among the major socioeconomic positive points included, the generation of employment and income, resulting from increased foreign exports and possibility to complement the growing energy demand, depend on oil and natural gas (Sundfeld and Machado, 2011).
With the expansion of sugarcane (Saccharum spp.) to new agricultural frontiers, there has been increased disease problems in the culture, among which can be emphasized the deleterious action of nematodes (Vianna and Sentelhas, 2014).Dinardo-Miranda and Menegatti (2003), estimate that in the culture of sugarcane damage caused by nematodes there have been about 20% reduction of crop production.Furthermore, the presence of nematodes in cultivated areas may prevent the use of such areas to new crops, thus making it uneconomical operation for certain crops in infested areas (Dinardo-Miranda, 2005).Meloidogyne sp. is known to root-knot nematodes, because of symptoms found in roots parasitized.Thus, the display of galls on roots is an indication of the presence of nematodes in the area (Dinardo-Miranda, 2005).Pratylenchus zeae (Graham) do not cause galls on the roots, but they are responsible for injuries resulting from the invasion of the cortical parenchyma of roots, producing extensive necrotic areas and causing serious economic losses (Vasconcelos and Dinardo-Miranda, 2011).
The control principle of immunization, by use of resistant or tolerant varieties is the method of controlling nematodes most practical and economical, however, they are not always having these characteristics (Silva et al., 2012).The use of systemic nematicides has been recommended by many researchers, and studies in which nematicides is applied at planting have found that these products contributes to productivity increases in soil infested with nematodes (Barros et al., 2006).Because the methods based on the use of chemical nematicides in soil are associated with environmental and social impacts of development, research with biocontrol agents have acquired greater importance (Qiuhong et al., 2006).In this context, rhizosphere microorganisms, known as rhizobacteria, has been able to promote substantial protection to nematoses (Siddiqui et al., 2001), especially Bacillus subtilis, as already reported at antagonist of root knot nematode (Li et al., 2005).
The effectiveness of biological control is directly related to the establishment of the antagonist on site.It has been reported that after the introduction of B. subtilis, there is a decline in the number of cells and spores with the passage of time (Adam et al., 2014), however Tokuda et al. (1995) stated that the introduction of cells in the soil results in the formation of spores to ensure the survival of the bacteria over long periods.It can be also emphasized that in perennial crops there are indications that the use of antagonists to the action of parasitism leads to more promising results because the establishment of the antagonist is facilitated and can suppress the survival of the pathogen as well as infection (Bettiol, 1991).In this sense, the aim of this study was to test the hypothesis that the efficacy and persistence of biological control (B.subtilis) is equivalent to the chemical control regarding the reduction of community Meloidogyne spp.and P. zeae in the soil.

MATERIALS AND METHODS
The experiment was conducted in greenhouse during the year 2009 using soil collected in the commercial area of cultive sugarcane in Morgado et al. 4491 Quatá, State of São Paulo.The area naturally infested with nematodes, with predominance species of the Meloidogyne incognita (Kofoid and White) Chitwood, Meloidogyne javanica (Treub) Chitwood and Pratylenchus zeae (Graham).The collected soil was packed in ceramic vessels with a capacity of 15 kg of soil.
The determinations of the initial populations (Pi) of the nematode were performed by soil sampling, before application of the treatments in the soil.The variety of sugarcane used was RB72454, susceptible to the action of nematodes (Marin, 2012).
The following treatments were conducted: control, B. subtilis (AP-3), B. subtilis (PRBS-1) and chemical nematicide (carbofuran).B. subtilis strains used in this study originate from soil under cultivation of soybean (Araujo et al., 2005).The experimental design was a randomized block design with four treatments and five replications.The treatments were applied in the furrow, effective with 40 cm long and 10 cm deep in the soil surface.Treatments with B. subtilis were applied at doses of 20 L ha -1 of cell suspension (1.2 ml per pot) and treatment with carbofuran was applied in the dose of 2 L ha -1 of active ingredient (0.11 ml per pot).Planting of sugarcane with two gems was done after this.
The suspension of vegetative cells of B. subtilis was produced in the laboratory.After growth of bacteria in tubes containing nutrient agar for four days, distilled water was added and a sweep performed after homogenization of cells were collected from the same resulting in a suspension that had a final concentration of 1.0 × 10 9 unit forms of colony (ufc) determined by the dilution method and plating on nutrient agar.Treatment with carbofuran was used with commercial product Furadan ® 350SC.
During the experiment, periodical sampling soil for extraction and quantification of nematodes in the laboratory according to the method of Coolen and D'Herde (1972) was carried out.To collect the Soil was collected in pots using a cylindrical probe dial, 15 cm depth was added to 10 cm of the stem of the plant at three equidistant points, and then the three sub-samples were homogenized for final collection of 100 cm 3 of soil.The evaluations were then based on nematode population densities in soil at 15, 30, 45, 60 and 75 days, respectively after planting and reproduction factor (RF = Pf / Pi) of the nematode studied as described in Barros et al. (2006).
At 75 days after planting, the plants were harvested and the roots were washed and separated from shoots with subsequent drying for 6 h at room temperature in shade.The roots were then weighed and then crushed in a blender for extraction of nematodes in accordance with the method cited above.Data were statistically analyzed using the Anova and Skott-Knott test (5%) for comparing averages.

RESULTS AND DISCUSSION
According to the results found monitoring Meloidogyne spp. in the soil there was significant reductions of the parasite from the second evaluation performed at 30 DAP (days after planting) in the treatments with the use of carbofuran and B. subtilis (Figure 1).The largest number of juveniles of Meloidogyne spp.found in soil was detected in the control treatment to assess the 30 DAP, while the treatments with carbofuran and B. subtilis was the total elimination of the parasite demonstrated in the final assessment performed at 75 DAP.Khan et al.  (2005), worked on assessing the population of M.
incognita in the soil, subjected to treatment with antagonists and biological nematicide, and observed reduction of the parasite population compared to the control, but there was no decrease in the population of the parasite in the soil in four consecutive evaluations held for 120 days.
The number of P. zeae in the soil, shown in Figure 2, express similar situation found in the evaluation of Meloidogyne spp. that is, from the evaluation 30 dap  were decreasing population of the parasite in soil treatments with B. subtilis and carbofuran.However, the control treatment recorded significant increases in nematode populations in all the tests conducted.In Table 1, it is observed that the soil nematicide treatments with B. subtilis provided lower reproduction factor (RF) of nematodes with greater emphasis on the reduction of RF in P. zeae.The FR below 1.0 indicates that the biological and chemical treatments reduced the nematode population in the soil.
Figure 3 shows the final results of the nematode population in the roots of sugarcane after 75 days of planting.It was found that P. zeae was present in the roots in greater number than the species of Meloidogyne spp.Reinforce indicating that this parasite causes various lesions resulting from their presence in the root tissue.
Both the evaluation of Meloidogyne spp.for the evaluation of P. zeae was found significant reduction of the presence of nematodes in the roots of sugarcane in the application of chemical and biological control.
The treatments with strains of B. subtilis (AP-3 and PRBS-1) resulted in significant reduction in infestation of nematodes in five evaluations in the soil and in the final assessment in roots of sugarcane.This performance was similar to that found in the control treatment with carbofuran (Figure 3).The result confirms the effectiveness of the action provided by nematicidal B. subtilis, described in the control of root-knot nematode in tomato (Araujo and Marchesi, 2009).The persistence of the control of nematode found in reviews suggests that rhizobacteria interferes with nematode reproduction, which was confirmed during the conduct of the experiment.Similar results were found by Sturz and Kimpinski (2004), which studied the effect of endophytic bacteria on the population density of Pratylenchus penetrans in potatoes found that the reduction of nematode population persisted for 14 weeks.
In this study, there was suppression of nematode parasites of sugarcane (Meloidogyne spp.and P. zeae) provided by chemical and biological treatments in the soil.The mode of action of the nematicide carbofuran occurs after absorption by the roots of the molecule with activation process that alters the composition of the substance responsible for the attraction of nematodes to the roots (Sikora, 1992).On the other hand, few studies were conducted in order to discover the mode action of B. subtilis on nematodes in soil.Some indications of mechanisms of control provided by B. subtilis on nematodes include the production of metabolites which reduces the outbreak and attraction to host-specific root exudates (Araujo et al., 2002); induction of systemic resistance mediated by bacteria in soil (Enebak and Carey, 2000); production of proteases that degrade the nematode cuticle and interfere with the cycle of the parasite (Lian et al., 2007), and that the nematodes and their eggs can serve as a food source for the bacteria (Khan et al., 2005).The different modes of action already described to elucidate the phenomenon of nematode control by B. subtilis may have contributed towards the suppressive effect of durability found in this study.B. subtilis can be useful in the future for use in biological control of nematodes in field conditions, as found in this study the efficacy and persistence of nematode control in sugarcane similar to that found with the use of conventional nematicide.

Conclusion
The hypothesis that B. subtilis could act effectively in control of nematodes (Meloidogyne spp., and Pratylenchus zeae), evaluated in this study was confirmed.It was also demonstrated that the persistence of suppressive action on nematodes provided by the treatments with B. subtilis on the ground, in a manner equivalent to that found by treatment with the use of conventional nematicide.

Figure 1 .Figure 1 .
Figure 1.Effect of carbofuran and Bacillus subtilis (AP-3 and PRBS-1) of the nematodes (Meloydogine spp.)At various times after planting.Letters same about the columns in each evaluation period are not statistically different at 5%

Figure 2 .Figure 2 .
Figure 2. Effect of Furadan and Bacillus subtilis (AP-3 and PRBS-1) on the nematode (Pratylenchus zeae) at various times after planting.Letters iguas about the columns in each evaluation period do not differ statistically at 5%. (Dap = days after planting).

Figure 3 .Figure 3 .
Figure 3. Number of Meloidogyne spp.and Pratylenchus zeae in roots of sugarcane at 75 days after planting in the presence of two strains of B. subtilis (AP-3 and PRBS-1) and carbofuran.Letters same about the columns in each evaluation period do not differ statistically at 5%.

Table 1 .
Reproduction factor (FR) of Meloidogyne spp.and Pratylenchus zeae in soil under cultivation of sugarcane for 75 days in a greenhouse after treatment with carbofuran and Bacillus subtilis (AP-3 and PRBS-1).

Number of juveniles 100 cm³ soil -1 Meloidogyne spp.
FR = Average P75/Pi-resulting from FR calculated for each repetition using the original data of populations.Means followed by letters, lowercase and uppercase letters in the column on the line, do not differ by the Scott Knott test at 5% probability. *