In vitro antagonistic activity of Bacillus subtilis strains isolated from soils of the Yucatan Peninsula against Macrophomina phaseolina and Meloidogyne incognita Actividad antagónica in vitro de cepas de Bacillus subtilis aisladas de suelos de la Península de Yucatán contra Macrophomina phaseoli

Instituto Tecnológico de Conkal, Km. 16.3 antigua carretera Mérida-Motul, Conkal, Yucatán. C.P. 97345. Tel. 52 999 912 4135. Address Correspondence to: Dr. Jesse Alberto Pacheco Aguirre; e-mail: jessep2010@gmail.com Recibido / Received 19.IX.2013. Aceptado / Accepted 26.X.2013. Resumen. La actividad antagónica de las cepas de Bacillus subtilis nativas de los suelos de la península de Yucatán fueron evaluadas en dos patógenos de suelo. Bacillus subtilis cbck36 y cbrf24 causaron la inhibición de más de 60% de crecimiento en la colonia de Machophomina phaseolina. El filtrado libre de células de B. subtilis cbr24 fue activo contra el segundo estado juvenil ( J2) de Meloidogyne incognita (CL50 25,8% v/v).


INTRODUCTION
Soil-borne pathogens such as fungi and nematodes reduce significantly fruit yield in a wide variety of economically important crops; yield losses range from 7 to 15% per year, equivalent worldwide to $100 billion approximately (Raaijmakers et al., 2009).In this context, the fungus Macrophomina phaseolina causes dry root rot, stem canker and stalk rot in approximately 500 plant species (Muñoz et al., 2005).The nematode Meloidogyne incognita causes root damage by direct feeding and establishment, which in turn leads to plant wilting and chlorosis in approximately 3000 plant species (Castagnone, 2002;Rahman, 2005).Chemical control using synthetic fungicides or nematicides is the most used control strategy (Parra & Ristaino, 2001); however, use of these synthetic products has caused various problems due to environmental pollution, human toxicity, as well as resistance of certain pathogens (Hernández et al., 2005).An alternative to reduce the effect of these plant pathogens is the use of antagonistic microorganisms, such as species of the genus Bacillus (Sid et al., 2003;Schisler et al., 2004).Bacillus subtilis is a bacterium that has been widely used as antagonistic for soil-borne pathogens.This bacterium produces hydrolytic enzymes such as glucanases or proteases and the antibiotic lipopeptides surfactin, fengycin, and/or iturin A, capable of acting against fungi and nematodes (Cazorla et al., 2007;Knaak et al., 2007;Snook et al., 2009).The present study was carried out to evaluate the in vitro effects of native B. subtilis on M. phaseolina and M. incognita.

MATERIALS AND METHODS
Bacillus subtilis strains were isolated from soil samples from the Yucatan peninsula.This region has calcareous soils, and a sub-humid climate with summer rains.Temperatures range from 21 °C to 34 °C throughout the year, and relative humidity ranges from 61% to 83%.Rainfall varies from 1037 mm to 1213 mm per year.From January to December 2012, twenty five soil samples were taken at 10 cm depth, near plants´ rhizospheres in different sites of the Yucatan Peninsula (Conkal, Cansahcab, Mesatunich, Muna and Calakmul).One gram of soil samples was mixed with 10 mL of sterile water under intensively stirring for 1 min at 25 °C.The samples were pasteurized (80 °C/30 min).Twenty μL of pasteurized samples were diluted in sterile water (1:10, v/v) (Escobar et al., 2004).The bacteria isolated in solution were carried off on nutrient agar.A preliminary screening using the direct confrontation technique allowed to evaluate the antagonistic activity of 25 B. subtilis strains against M. phaseolina.Those strains which caused mycelial growth inhibition above 30% were selected.This yielded a total of seven active strains: cbcc2, cbrf24, cbmn22, cbck47, cbck36, cbmt2 and cbmt51.The molecular identification of the isolates was performed by amplification and sequencing of the 16S rRNA genes by PCR (Geetha et al., 2007).Macrophomina phaseolina (Tassi) was donated by Dr. Mario Ramírez-Lepe from the Veracruz Technological Institute, located in Veracruz, México.The antifungal activity on M. phaseolina was performed by direct confrontation on Potato Dextose Agar (PDA), using a 4-mm diameter PDA disc of fungal growth and 1 x 10 7 CFU/mL bacterial suspensions.Six days after inoculation, the growth inhibition zone and the percent inhibition of fungal colony growth were measured (Rodas et al., 2009).Meloidogyne incognita was obtained from a stock colony maintained in tomato (Lycopersicon esculentum Mill.) in a greenhouse, at the Conkal Technological Institute, located in Yucatan, México.The nematicidal activity of the cell-free culture filtrates on second-stage juveniles ( J2) of M. incognita was evaluated as described by Cristobal et al. (2006).Cell-free filtrates from bacteria cultivated in Nutrient Broth (72 h, 29 °C, and 200 rpm) were obtained by centrifugation (3000 rpm, 20 minutes) and filtering through a Millipore filter of 0.22 μm.Bacterial filtrates were diluted in distillated water (25%, 50% and 75%) and poured in Syracuse dishes, where J2 were placed (100 J2 per mL filtrate) to calculate the median lethal concentration (LC50) after 9 h exposure.Mortality of nematodes was recorded after 3, 6, 9 and 24 h of exposure to calculate the median lethal time (LT50) at a concentration of 50% (v/v).For analysis of variance, data were transformed to arcsin [y = arcsin (sqrt y/100)].Multiple mean comparisons (Tukey, p=0.05) and calculation of LC 50 and LT 50 by probit analysis were performed using the Statgraphics Centurion Software (15.2.06 version).

DISCUSSION
The B. subtilis strains cbck36 and cbrf24 (evaluated in this study) showed the highest antagonistic activity on M. phaseolina and M. incognita, respectively.The presence of growth inhibition zones in the antifungal bioassays, and the activity of the cell-free culture filtrates on M. incognita, suggest that the antagonistic activity of these strains might be in part due to the production of antimicrobial metabolites.Previous works have shown that B. subtilis produces at least five different antimicrobial compounds: subtillin, bacitracin, bacillin, subtenolin, and bacilonycin (Killani et al. 2011).We also observed that some strains, like cbcc2 and cbmt2 inhibited mycelial growth of M. phaseolina by direct competition, as the bacterial colonies were able to growth radially on the PDA.
The activity of the cell-free culture filtrate of cbrf24 on J2 M. incognita suggests that this strain might be able to produce toxic metabolites against this nematode.The presence of toxic components in cell-free culture filtrates of Bacillus spp. on nematode immobility and subsequent mortality has been previously reported (Caneiro et al., 1998).
We found that the cbrf24 strain of B. subtilis showed a wider antagonistic activity than the cbck36 strain of the same species on two soil-borne fungi pathogens, M. phaseolina and M. incognita.This strain might be a good candidate to develop a microbial based biopesticide.Additional research is required to identify the active compounds excreted by this bacterium during growth.

Table 1 .
Bacillus subtilis on Macrophomina phaseolina and Meloidogyne incognita Mean values (± standard error) of inhibition of fungal colony growth and growth inhibition zone caused by B. subtilis strains on M. phaseolina.Valores medios (± error estándar) de inhibición fúngica del crecimiento de colonias y zonas de inhibición causadas por las cepas de B. subtilis sobre M. phaseolina.