Abstract: The isolation of Bacillus strains with antifungal activity is important to carry out tests to protect economically important crops. We determined the in vitro activity of 13 native strains of Bacillus sp. isolated from soil against Macrophomina pasheolina using a dual assay in nutrient agar. All the strains showed inhibition of radial growth of M. phaseolina from 31-80%, with strains LUM B01 and B04 in a 80.8 and 75.9%, respectively with the highest antifungal activity. The crude extract of the strain of chitinases LUMB0 04 inhibited the growth of M. phaseolina by 30%. The strains of chitinolytic Bacillus sp. isolated from soils showed antifungal activity and have the potential to be used in biological control of M. phaseolina.

INTRODUCTION

Macrophomina phaseolina is the causal agent of stem rot in various crops such as beans and soya bean. Phytopathogenic fungi are agents that cause economic losses in agriculture and to control these diseases is the use of common chemical pesticides, however has seen the development of fungicide resistance by fungal pathogens (Ishii, 2004), so antagonistic microorganisms such as bacteria and fungi are an alternative for controlling these pathogens.

Antagonism acts on antibiosis, competition or parasitism. Parasitism involves the production of hydrolytic enzymes as chitinases and β-1-3 glucanase (Woo et al., 2002; Compant et al., 2005; Prapagdee et al., 2008). The chitinases have an effect on the germination of conidia (Huang et al., 2005; Huang and Chen, 2008) and degrade the cell wall of pathogenic fungi (Gupta et al., 1995). Production of chitinolytic enzymes by fermentation can be performed using ground shrimp wastes (Reyes-Ramirez et al., 2004). Bacteria of the genus Bacillus has showed antifungal activity against phytopathogenic microorganisms (Cho et al., 2003; Feio et al., 2004; Huang et al., 2005; Hu et al., 2008). Isolation of strains of the genera Bacillus can be made directly from the soil and the activity of chitinases production can be determined by testing hydrolysis in medium with chitin (Rodas-Junco et al., 2009). These bacteria have the advantage of being already adapted to the environment where they can be applied as biological control. Studies of in vitro antifungal activity of the dual rate should be measured in a culture medium that allows growth of both types of microorganisms. For studies of in vitro antagonism on bacteria against phytopathogenic fungi is common to use the medium and Potato Dextrose Agar (PDA) (Lokesha and Benagi, 2007; Hu et al., 2008) that favors the growth of fungus. These types of tests allow the screening of potentially beneficial organisms to control plant pathogenic fungi. The selection of microorganisms with the inhibition of Macrophomina could be useful for evaluating the potential in greenhouse and/or open field production.

The aim of this study was to evaluate the antifungal activity of native strains of Bacillus sp. isolated from soil against M. phaseolina in dual bioassays using Nutritive Agar (NA), as this medium allowed growth of both types of microorganisms.

MATERIALS AND METHODS

Microorganisms: We used 13 native strains of Bacillus sp. presenting activity of chitinases (Rodas-Junco et al., 2009). These were isolated from different types of cultivated and uncultivated soils. The strains were maintained on NA at 4°C and were activated by seeding Petri dishes with nutrient agar and incubated 30°C for 24-48 h. The strain of M. phaseolina was kindly donated by Dr. Mario Ramirez-Lepe from the Technological Institute of Veracruz (ITV, Mexico) and was maintained on PDA and then growth at 28°C, 72 h and stored at 4°C.

Dual test: A dual type bioassay was performed (Huang et al., 2005; Lokesha and Benagi, 2007) to determine the percentage of growth inhibition of M. phaseolina by strains of Bacillus sp. Seeding of the strains of Bacillus sp. and M. phaseolina was performed in a Petri plate with NA at 3 cm between them and the strains of Bacillus sp. and M. phaseolina in an individual as control, were incubated at 28°C for 6 days, the radial growth of microorganisms was followed and the inhibition percentage was calculated according to the formula reported by Lokesha and Benagi (2007).

Chitinases production: To obtain crude extract of chitinases, crushed shrimp shell was used as a source of chitin at 6% in distilled water, fermentation was performed at 28-30°C and 250 rpm for 120 h (Reyes-Ramirez et al., 2004), the crude extract was recovered by centrifugation at 1972xg, 5°C for 15 min. The enzyme activity was determined according to Monreal and Reese (1969) for the release of N-Acetyl Glucosamine (NAG) using colloidal chitin as substrate. One Unit of chitinases (U) was defined as the amount of enzyme that catalyzes the release of 1 mg of NAG/h at 50°C. The crude extract was sterilized with a 0.22 μm Millipore membrane (Millipore Corporation, Bedford, USA) to obtain a crude extract of free cells.

Effect of chitinase on Macrophomina phaseolina: The crude extract was adjusted to pH 6.0, to avoid growth inhibition by an alkaline pH. The crude extract was used to 10% on PDA, added to the medium before solidifying and dispensed into a petri plate was inoculated with a 5 mm disk of mycelium of M. phaseolina in the center and radial growth was recorded for 6 days. Phosphate buffer pH 6.0 was used as control. Five repetitions were performed. The results were analyzed by an Analysis of Variance (ANOVA) and Tukey´s test (p≤0.05) using SPSS 15.0.1 for Windows (Lead Technologies, Inc. USA).

RESULTS AND DISCUSSION

The strains of Bacillus sp. used in this study were previously isolated from different soil types, cultivated and uncultivated in the district of Juquila, Oaxaca, Mexico. The selected strains produce chitinases and proteases (Rodas-Junco et al., 2009). Isolates of Bacillus sp. were made by Gram staining, catalase test and formation of endospores as confirmatory evidence of the genus Bacillus and the production of chitinases by the hydrolysis of halo formation in minimal medium with colloidal chitin according to Rodas-Junco et al. (2009). The dual test was conducted in petri plates with NA medium, the medium of choice is not the usual for the growth of fungi (PDA), however, showed a radial growth of Macrophomina of 54 mm to 120 h, similar to that achieved in the PDA at 72 h, despite a slower growth observed, was sufficient for both types of microorganisms and dual test (Fig. 1). The radial growth of microorganisms and the percentage of inhibition calculated in all the strains tested showed a decrease in growth of M. phaseolina and was observed a higher percentage of inhibition in the strains LUM B01 with a 80.8±6.5%; LUM B04 (75.9±3.8%); LUM B57 (68.5±1.5%); LUM B65 (66.3±1.5%) and LUM B19 (65.9±7.8%).

There was no significant difference (p≤0.05) between these strains (Fig. 2). All strains of the used Bacillus sp. showed a percentage inhibition >50% (range 54-80%), except for strain LUM B15 with a 31.1±1.5%. Values close to 75% inhibition of growth of Macrophomina by Bacillus subtilis were observed by Lokesha and Benagi (2007) using a PDA culture medium.

Fig. 1:	Antifungal activity of native strains of Bacillus (a) LUM B01 and (b) LUM B04 on NA plates against M. phaseolina. MP: M. pahaseolina; Bc: Bacillus sp.

These values are comparable to those observed in this research by strains LUM-B01 and B04. We observed a more rapid radial growth of the strain LUM B01, so in the dual test, reached the fungus at 72 h (Fig. 1), this could explain the higher percentage of inhibition of this strain due to competition between the two organisms for the available space in the petri plate. Once, they reached the contact with the fungus strain they followed the bacterial growth on the fungus inhibiting further growth of Macrophomina, possibly in a process of parasitism. This was observed in strains of Bacillus sp. that grew to reach the fungus. We observed the growth inhibition of M. phaseolina without contact with the bacterial strains (Fig. 1), this could be due to the production of hydrolytic enzymes or antibiotics extracellular compounds, which are disseminated through the media. In B. subtilis and B. pumilus have been identified antibiotics with antifungal activity (Leifert et al., 1995). To test the effect of hydrolytic enzymes on the growth of Macrophomina, we used a crude extract with activity of chitinases produced by fermentation using as a source of ground shrimp shell chitin. For the production of chitinases strains LUM B01 and B04 were used, for presenting the highest percentage of in vitro inhibition of M. phaseolina. The fermentation was conducted at 120 h as it is the maximum time reported for production of chitinases of Bacillus strains using shrimp wastes (Reyes-Ramirez et al., 2004), both strains were producing chitinases (Fig. 3). The production of chitinases was significantly higher in the strain LUM B04 at 96 h, but at 120 h of fermentation there is no significant difference being 0.435±0.035 U mL^-1 for LUM B01 and 0575±0.007 U mL^-1 for LUM B04. Possibly this is due to a decrease in the production of chitinases around 102 h due to the presence of proteases in the medium (Reyes-Ramirez et al., 2004). To determine the effect of chitinases on the growth of M. phaseolina, crude extract of the strain LUM B04 of 120 h of fermentation was used, because it presented the highest production of chitinases. There was a significant decrease of radial growth from the 48 h, the crude extract showed a decrease of nearly 30% compared with the control (Fig. 4). A nearly 100% decrease reported in Sclerotium rolfsii using a crude extract of chitinases concentrated by ultrafiltration, the difference in the rate of growth varies according to different types of species are reported from 18-90% and the concentration of activity enzyme used (Reyes-Ramirez et al., 2004). The slowdown in the growth of M. phaseolina is due to the presence of chitinases in the environment, however there may be involvement of other enzymes such as β-1-3 glucanase, as well as other molecules such as antibiotics that may involved in the growth inhibition of phytopathogenic fungi (Leifert et al., 1995). Chitinolytic microorganisms are considered as having the potential for biocontrol of phytopathogenic soil for their antagonistic ability.

Fig. 2:	In vitro inhibition of growth of M. phaseolina by Bacillus sp.

Fig. 3:	Chitinolytic activity of LUM B01 and LUM B04 growing on shrimp wastes

Fig. 4:	Effect of crude chitinases extract from Bacillus sp. LUMB 04 on the growth of M. phaseolina on PDA plates

CONCLUSION

Dual assay method allows determining antifungal activity of the native strains of Bacillus sp., showing higher activity of strains LUM B01 and B04. Hydrolytic enzymes affect the growth of M. phaseolina. The native strains of Bacillus sp. LUM B01 and B04 are chitinolytic microorganisms with antifungal activity, could be evaluated in vivo to test the potential to be used in biological control of plant pathogenic fungi.

ACKNOWLEDGEMENTS

This research was supported by Universidad del Mar (2IR0903). We thank Mario Ramirez-Lepe, UNIDA-ITV, for providing the strain of M. phaseolina and acknowledge Addy Guzman and Gabriela Ruelas for technical collaboration.