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In vitro growth inhibition of Curvularia gudauskasii by Bacillus subtilis

In vitro growth inhibition of Curvularia gudauskasii by Bacillus subtilis Ann Microbiol (2012) 62:545–551 DOI 10.1007/s13213-011-0290-x ORIGINAL ARTICLE In vitro growth inhibition of Curvularia gudauskasii by Bacillus subtilis Teresa Orberá Ratón & Zenia González Giro & Manuel Serrat Díaz & Suyén Rodríguez Pérez Received: 25 January 2011 /Accepted: 30 May 2011 /Published online: 30 June 2011 Springer-Verlag and the University of Milan 2011 Abstract Five rhizosphere Bacillus subtilis isolates were In Cuba, Curvularia plant pathogen species are associ- tested for in vitro antagonism against a sugarcane seedling ated with sugarcane seedlings, producing root rottenness pathogen strain of Curvularia gudauskasii. The isolate B. and malformations affecting fertility and seedbed efficiency subtilis SR/B-16 was selected because it permanently (Alfonso et al. 1990). The most common species, Curvu- inhibits 71% of fungal growth. Microscopic analysis of laria lunata, Curvularia senegalensis and Curvularia the antagonistic bacteria effects on C. gudauskasii revealed gudauskasii affect seed bank yields by up to 30% (Alfonso swelling and bulbous hyphae, vacuolated cytoplasm and no et al. 1990; Chinea 2005). Treatment of this plant disease is spore formation. The analysis of culture filtrates of SR/B- usually by chemical fungicides. Some attempts at selecting 16 collected from stationary phase showed that they are biological control agents (BCA) against Curvularia phyto- responsible for the antifungal effects and for the abnormal pathogen species have revealed their in vitro susceptibility shapes observed. However, bacterial culture efficiency is to Trichoderma viridae, Trichoderma harzianum and higher. The organic concentrated fraction of the extracts Trichoderma lignorum (Alfonso et al. 1990). Also, Pseu- caused the same morphological changes as those caused by domonas and Bacillus strains have been reported to have antifungal lipopeptides secreted by Bacillus species. The inhibitory activity against C. lunata in dual culture assays strain SR/B-16 can be used for the formulation of bioactive (Alfonso and Villa 2002; Basha and Ulaganathan 2002; compounds for the treatment of C. gudauskasii diseases on Tendulkar et al. 2007). sugarcane seedlings. Bacillus and its relatives are attractive candidates for bio-controlling plant pathogens because they produce . . . Keywords Bacillus subtilis Culture filtrate Antifungal extracellular metabolites such as antibiotics lipopeptides . . Curvularia gudauskasii Hyphal abnormality Biocontrol and hydrolytic enzymes, which play an important role in the direct antagonism of plant disease fungi (Kildea et al. 2008;Yangetal. 2008; Saidi et al. 2009). Their Introduction interaction with filamentous fungi induces hyphae mal- formations related to their biological control mechanisms. Numerous plant species are affected by Curvularia plant The predominant morphological alterations are swelling pathogen fungi, mainly under climatic stress periods with and bulbous formation, hyphae distortion, cell wall high temperatures and environmental humidity. Curvularia darkness, cytoplasm aggregation, inhibition of spore ger- phytopathogen species produce spots in grains and seeds, as mination and hyphae tip rupture (Rahman et al. 2007; well as damage to plant leaves (Barrios and Pérez 2005). Prapagdee et al. 2008). In a previous study, five Bacillus subtilis rhizosphere : : : T. Orberá Ratón (*) Z. G. Giro M. Serrat Díaz S. R. Pérez strains exhibited a marked inhibitory effect on the plant Centro de Estudios de Biotecnología Industrial, pathogen fungi Curvularia lunata, Fusarium oxysporum Facultad de Ciencias Naturales, Universidad de Oriente, and Colletotrichum sp. (Orberá et al. 2009). This research Avenida Patricio Lumumba S/N, was aimed at evaluating if the bioactive compounds 90500, Santiago de Cuba, Cuba secreted by those B. subtilis antagonistic isolates are able e-mail: torbera@cebi.uo.edu.cu 546 Ann Microbiol (2012) 62:545–551 to generate morphological alterations related to the in vitro growth inhibition percentage (RGIP) based on the following growth inhibition of a Curvularia gudauskasii strain formula: causing damage to sugarcane seedlings. R1  R2 RGIP ¼  100% ð1Þ R1 Materials and methods where, Microorganisms R1 Radial growth of C. gudauskasii in the control plate R2 Radial growth of C. gudauskasii interacting with the Bacteria antagonistic B. subtilis strain Five aerobic endospore-forming bacterial isolates with antagonistic properties against plant pathogenic fungi were used (Orberá et al. 2009). The strains were previously Hyphal morphology identified as Bacillus subtilis and the 16S rRNA gene partial sequences accession numbers, deposited at the Following positive antibiosis test results, hyphal strands National Centre for Biotechnology Information Database from the border of the fungal colony nearest to the growth are: SR/A-1 [HQ025915], SR/B-8 [HQ025916], SR/B-16 inhibition zone were removed smoothly using a sterile [HQ025917], SR/B-17 [HQ025918], and SR/A-17 needle and placed on a microscope slides with lactophenol [HQ025919] (NCBI, http://www.ncbi.nlm.nih.gov). 28.5% (BDH Chemicals, Poole, UK) and examined under a light microscope (40×), looking for hyphal abnormalities. Fungi Microphotographs were taken using a digital camera (Canon Power Shot A640; Rahman et al. 2007). A phytopathogen strain of Curvularia gudauskasii was provided by the Phytopathology Laboratory strain collec- Bacterial growth kinetics tion at the National Sugarcane Research Institute (INICA), Havana, Cuba. fresh bacterial culture of B. subtilis SR/B-16 from a NA The microorganisms were maintained at −20°C on glycerol, slant previously grown for 18 h at 30°C, was inoculated at the Industrial Biotechnology Study Centre Culture Collec- into a 250 mL Erlenmeyer flask containing NB medium. tion (CCEBI) [http://www.aam.org.ar/cultivos_microbianos. The culture was incubated at 30°C for 12 h and shaken shtml]. Bacteria were subcultivated on nutrient agar vigorously at 150 rpm. For growth curve estimation, 1 mL (NA, Biolife Italiana, Milan, Italy) and nutrient broth from the pre-inoculum was inoculated into a 500-mL (NB, Biolife Italiana). Fungi were grown on potato Erlenmeyer flask containing 100 mL NB. The culture was dextrose agar (PDA, UNI–CHEM Chemical Reagents; then incubated for 72 h at 30°C. Cellular growth was http://www.unichem-chemicals.com/). determined spectrophotometrically by measuring changes in optical density (OD) with time at 620 nm. Bacillus subtilis strains with antifungal activity against Curvularia gudauskasii Characterization of bacterial culture filtrates The dual culture assay was performed in Petri dishes Bacterial culture was transferred to NB and incubated for containing PDA medium. A 5-mm diameter mycelial 6 and 21 h, while shaking vigorously at 150 rpm at 34°C. plug taken from a 5-day-old culture of C. gudauskasii Cells were removed by centrifugation at 5,000 rpm at 10°C was transferred to the center of the Petri dish. One for 10 min. The supernatant collected was filtered through a milliliter of each Bacillus subtilis strain suspension 0.2 μm pore size Millipore membrane. The protein and 6 −1 (10 CFU mL ) was inoculated at an equidistant point carbohydrate content, as well as the qualitative presence of (20 mm) from the Petri dish center, which was inoculated lipids were determined on the culture filtrates. Protein with the mycelial plug. The plates were incubated in the content was determined following the Lowry technique, dark at 30°C. Trials were done in triplicate and a control using a solution of bovine serum albumin (BSA; BDH, was prepared with the fungal culture without bacterial Poole, UK) as standard (Lowry et al. 1951). The absor- inoculation. The appearance of a growth inhibition zone of bance was read at 650 nm. Soluble carbohydrates were the fungal colony was taken as a positive result (Muhammad determined using the phenol–sulfuric method (Dubois et al. and Amusa 2003). The inhibitory effect was assessed 1956). Spectrophotometer readings were taken at 490 nm. −1 following Ezziyani et al. (2004), by calculating the radial A glucose solution (UNICHEM, 1.00 g L ) was used as Ann Microbiol (2012) 62:545–551 547 standard. The qualitative presence of lipids was determined using the Sudan III coloring technique (Díaz et al. 1989). A test tube with sterile medium was used as a negative control. Concentration of free-cell extracts Sterile extracts were separated with chloroform: methanol mixture (2:1) according to the Folch method (Folch et al. 1957). The organic extract was concentrated in vacuo, yielding a brownish gummy residue, which was suspended in 5 mL phosphate buffer at pH 6.5. The soluble phase was Fig. 1 In vitro radial growth percentage inhibition (RGPI) of kept at 4°C to be used later. Curvularia gudauskasii by Bacillus subtilis isolates. Different lower case letters indicate statistical differences at P≤0.005 Antagonism test with free-cell extracts Antagonism tests with free-cell extracts were carried out using Curvularia gudauskasii mycelium alterations the plate diffusion technique described by Vignolo et al. upon interaction with Bacillus subtilis antagonistic strains (1993). Sterile filtrates collected at 6 and 21 h of incubation were evaluated, as well as the organic and inorganic fractions Dual culture on Petri dishes of B. subtilis strains SR/A-1, obtained from the 21-h extracts. Aliquots of 100 μLof the SR/B-8 and SR/B-16 with C. gudauskasii is shown in crude extract were deposited in sumps opened in the surface Fig. 2. A growth inhibition zone produced by bacteria of the solid medium surface on Petri dishes, previously extracellular metabolites was observed around the fungal inoculated with a mycelium plug. The plates were incubated mycelium. This zone was visible next to isolates SR/A-1, at 30°C for 7 days until the appearance of mycelium growth SR/B-17 and SR/A-1 from the 3rd to the 5th incubation inhibition zones. Hyphal abnormalities were studied as day, when the fungal growth restarted; however, the rate described (Rahman et al. 2007). was lower and a loss of the typical dark color of mature Curvularia colonies was observed (Fig. 2d). As Curvularia Statistical analysis pigmentation is attributed to the presence of asexual conidia, this loss of color indicated that antagonistic B. subtilis strains inhibited spore formation in this plant The means and standard deviations of the inhibition of radial growth were calculated. Data were analyzed by one- pathogen fungus. No mycelial growth was seen around way analysis of variance (ANOVA). Significant differences strains SR/B-8 and SR/B-16 after 15 incubation days. (P≤0.05) among the means were determined by Duncan’s Necrosis and darkening colour were also observed in the multiple range tests, using the software Statgraphics Plus 5.1 border of the colony nearest these isolates (Fig. 2d). B. for Windows (http://www.statgraphics.com/statgraphics_plus. subtilis SR/B-16 showed the strongest antifungal effects, htm). and the study was continued using only this strain and its extracellular metabolites. Microphotographs showed hyphal swelling, bulb formation, a granular cytoplasm with an Results intense vacuolization, and absence of conidia (Fig. 2e–f). In vitro growth inhibition of Curvularia gudauskasii Cell-free extract effects of SR/B-16 on Curvularia by Bacillus subtilis gudauskasii morphology Growth inhibition of C. gudauskasii by B. subtilis isolates The encounter of C. gudauskasii growing mycelium and is represented in Fig. 1. Bacteria showed values of RGIP of SR/B-16 sterile filtrates collected during its active growth, between 41 and 71%, corresponding to the isolates SR/A- did not show any alterations on hyphae and colony 17 and SR/B-16, respectively. Strains SR/B-8 and SR/B-16 morphology. However, fungal cultivation with the extracts had similar significantly higher inhibitory effects; however, obtained at stationary phase (21 hours) corroborated that the latter was the most efficient. All the strains were they contain the metabolites implicated in bacterial anti- classified as moderately toxic according to the scale used fungal activity (Fig. 3). The in vitro interaction of C. by Ros et al. (2008), which considered as toxic only those gudauskasii with the collected sterile extract of stationary isolates inhibiting fungal growth by 100%. phase B. subtilis SR/B-16 generated a mycelium growth 548 Ann Microbiol (2012) 62:545–551 Fig. 2 In vitro interaction effects of B. subtilis antagonistic strains and vacuolization, bulb formation, swelling, no conidia); g fungal colony C. gudauskasii (40× magnification). a Control plate; b,c control in interaction with SR/B-16 sterile extracts; h C. gudauskasii hyphae mycelium; d dual culture of C. gudauskasii and B. subtilis isolates; e,f interaction with SR/B-16 sterile extracts (arrows swelling, vacuoliza- C. gudauskasii hyphae encounter with B. subtilis SR/B-16 (arrows tion, no conidia). IZ Growth inhibition zone, CE cell free extracts inhibition zone (Fig. 2g). The corresponding hyphae showing that they were consumed as nutrients. The microphotographs showed swelling, intense vacuolization qualitative presence of lipids was determined only in and no conidia formation (Fig. 2h). extracts collected at stationary phase. Characteristics of SR/B-16 free-cell extracts Antifungal effects of concentrated extracts of SR/B-16 Protein and carbohydrate contents in the sterile extracts The cell-free extract of SR/B-16 strain was partitioned into collected at 6 and 21 incubation hours were determined aqueous and organic phases in order to evaluate their effects (Fig. 3). Total protein content was higher in exponential on fungal morphology. The in vitro interaction of the phase, and decreased when active growth ended. Carbohy- organic fraction of B. subtilis SR/B-16 sterile extracts with drates decreased along with bacterial growth kinetics, C. gudauskasii caused abnormal appearance of the fungal Ann Microbiol (2012) 62:545–551 549 lycopersici isolated from tomatoes cultivars by more than 50% (Saidi et al. 2009). In order to determine how rhizobacterial isolates affect fungal growth, the presence of hyphal malformations in C. gudauskasii colonies was investigated using a dual culture assay with B. subtilis SR/B-16. The bacterial strain and its sterile crude extracts showed the highest antagonistic activity. The swelling and the absence of conidia observed in the plant pathogen fungus under the light microscope are abnormalities caused by antagonistic bacteria and conven- tional antifungal compounds interacting with filamentous fungi. Swelling has been detected in the presence of Fig. 3 Bacillus subtilis SR/B-16 growth on nutrient broth (NB). quitinases enzymes and antibiotics such as iturins, sub- Protein and carbohydrate concentrations in the crude extracts during tilisins and surfactins (Tendulkar et al. 2007; Rahman et al. growth stages are indicated 2007; Prapagdee et al. 2008). Swelling is also related to the cells. Microphotographs (Fig. 4) show cell wall thickening, disorganization of cellular actin filaments produced by a reduction in hyphae diameter and the absence of asexual secondary metabolites from antagonistic rhizobacteriae conidia. The interaction of C. gudauskasii with the (Deora et al. 2010). The absence of conidia has been inorganic fraction did not generate mycelium alterations. reported as one of the malformations generated by antifungal isolates of Paenibacillus lentimorbus, Burkhol- deria cepacia and Streptomyces hygroscopicus and their Discussion sterile filtrates (Chen et al. 2003; Rahman et al. 2007; Prapagdee et al. 2008). Both swelling and the absence of The present study explored, for the first time, the potential conidia were observed on Neurospora crassa grown in the use of Bacillus subtilis rhizosphere isolates for the presence of conventional chemical fungicides (Pereira and biological control of the sugarcane seedling pathogenic Said 2009). fungus Curvularia gudauskasii. In vitro fungal growth The necrosis observed in the mycelium border at the inhibition values coincided with previous results shown by interaction zone with the strains SR/B-8 and SR/B-16 is other researchers. In Cuba, inhibition of Curvularia probably related to fungal growth inhibition. It is well known phytopathogenic species by B. subtilis ATCC 6633 with that alterations in the apex of hyphae of filamentous organisms radial growth inhibition has been reported to be between 66 affect major biological processes such as growth, reproduc- and 100% (Castellanos et al. 2002). Rahman et al. (2007) tion, morphogenesis, nutrient absorption and protein secretion isolated Pseudomonas aeruginosa and Burkholderia cepa- taking place in the tips (Pereira and Said 2009). cia strains that strongly inhibited the growth of Colleto- The morphological alterations in C. gudauskasii seen upon trichum gloeosporoides by an average of 74 and 68%, application of crude extracts of SR/B-16 are less severe than respectively. Numerous Bacillus species inhibited the in those seen in dual culture assay with bacterial colonies. This vitro growth of a Fusarium oxysporum f.sp. radicis- suggests that bacteria act through a combination of at least Fig. 4 Morphological abnormalities of C. gudauskasii mycelium in encounter with organic extract (arrows distortion, hialinization, bulbs interaction with the organic concentrate of B. subtilis SR/B-16 crude formation, absence of conidia) extract (40× magnification). a Control mycelium; b,c mycelium 550 Ann Microbiol (2012) 62:545–551 two biological control mechanisms: nutrient competition and iturins, surfactins, and fengicins, excreted as secondary antifungal metabolite secretion. A similar phenomenon was metabolites. All play different roles in the growth and produced by a B. subtilis strain with inhibitory effects on survival of Bacillus species in their natural habitat, Trichoderma species contaminating commercial mushroom rhizosphere colonization and their biological control efficacy production (Chittihunsa et al. 2007). In vitro dual culture (Arguelles-Arias et al. 2009). However, to confirm our betweenanantagonistic Bacillus lentimorbus isolate and suggestions regarding the nature of the antifungal com- Colletotrichum gloeosporoides showed that nutrient compe- pounds from B. subtilis SR/B-16, more studies, including tition at early stages and antibiosis at advanced stages are characterization and purification tests, mass spectra and responses to their stress interaction (Lee et al. 2005). This NMR, will be needed. result is invaluable during the selection of an efficient biological control agent (BCA). Antifungal metabolite secretion is known to offer more advantages than competi- Conclusions tion, but the synergistic effects in a BCA strain increase considerably its effectiveness as a plant disease treatment. B. subtilis rhizosphere isolates were evaluated for their Bacillus subtilis SR/B-16 developed diauxic growth in NB potential for biological control of the phytopathogen C. culture medium, which may be attributed to the initial gudauskasii. The wild type strain SR/B-16 inhibits the in hydrolysis of peptone, a simple carbon source present in the vitro growth of the plant pathogen fungus by more than culture medium. After that, SR/B-16 entered a second lag 70%, probably through a combination of two biological phase, probably developing physiological mechanisms to control mechanisms: nutrient competition and the excretion hydrolyze beef extract, a more complex carbon source present of antifungal lipopeptides as secondary metabolites. Sterile in NB, showing the wider potential of this strain to grow in a filtrate collected at stationary phase contains the bioactive variety of carbon sources. Both peptone and beef extract compounds responsible for the alterations in morphology supplied B. subtilis SR/B-16 with the nutrients required for and growth pattern, as well as the absence of conidia, which growth. In the technical sheet of NB culture medium from are related to fungal growth inhibition. Therefore, we Biolife Italiana (http://www.biolifeit.com/biolife/upload/file/ suggest that B. subtilis SR/B-16 may be used as an Schede/TS-401810.pdf), the culture medium is referred to as inoculant for the biological control of diseases caused by beef peptone media. Peptone hydrolysis by Bacillus strains C. gudauskasii in sugarcane seedbeds. has been reported by Vasileva-Tonkova et al. (2007). Acknowledgment The authors are grateful for the assistance of Juan The presence of proteins in the sterile extracts of SR/B- Alfonso Ayala Serrano, PhD from the Molecular Biology Institute 16 is attributed to the ability of Bacillus and its relatives to “Severo Ochoa”, with the molecular identification of the Bacillus excrete numerous enzymes, signals and antifungal peptides isolates used in this research. (Arguelles-Arias et al. 2009). The decrease in the final protein content may be provoked by the production of References bacterial extracellular proteases. Razo et al. (2008) reported maximum levels of proteases in a B. subtilis strain at the end of its active growth. Alfonso I, Villa P (2002) Evaluación de la cepa de Pseudomonas spp. The detection of proteins and lipids in SR/B-16 crude cepa PSS en el control de hongos fitopatógenos de las semillas agámicas y botánicas de la caña de azúcar. 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In vitro growth inhibition of Curvularia gudauskasii by Bacillus subtilis

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Springer Journals
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Copyright © 2011 by Springer-Verlag and the University of Milan
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Life Sciences; Microbiology; Microbial Genetics and Genomics; Microbial Ecology; Mycology; Medical Microbiology; Applied Microbiology
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10.1007/s13213-011-0290-x
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Abstract

Ann Microbiol (2012) 62:545–551 DOI 10.1007/s13213-011-0290-x ORIGINAL ARTICLE In vitro growth inhibition of Curvularia gudauskasii by Bacillus subtilis Teresa Orberá Ratón & Zenia González Giro & Manuel Serrat Díaz & Suyén Rodríguez Pérez Received: 25 January 2011 /Accepted: 30 May 2011 /Published online: 30 June 2011 Springer-Verlag and the University of Milan 2011 Abstract Five rhizosphere Bacillus subtilis isolates were In Cuba, Curvularia plant pathogen species are associ- tested for in vitro antagonism against a sugarcane seedling ated with sugarcane seedlings, producing root rottenness pathogen strain of Curvularia gudauskasii. The isolate B. and malformations affecting fertility and seedbed efficiency subtilis SR/B-16 was selected because it permanently (Alfonso et al. 1990). The most common species, Curvu- inhibits 71% of fungal growth. Microscopic analysis of laria lunata, Curvularia senegalensis and Curvularia the antagonistic bacteria effects on C. gudauskasii revealed gudauskasii affect seed bank yields by up to 30% (Alfonso swelling and bulbous hyphae, vacuolated cytoplasm and no et al. 1990; Chinea 2005). Treatment of this plant disease is spore formation. The analysis of culture filtrates of SR/B- usually by chemical fungicides. Some attempts at selecting 16 collected from stationary phase showed that they are biological control agents (BCA) against Curvularia phyto- responsible for the antifungal effects and for the abnormal pathogen species have revealed their in vitro susceptibility shapes observed. However, bacterial culture efficiency is to Trichoderma viridae, Trichoderma harzianum and higher. The organic concentrated fraction of the extracts Trichoderma lignorum (Alfonso et al. 1990). Also, Pseu- caused the same morphological changes as those caused by domonas and Bacillus strains have been reported to have antifungal lipopeptides secreted by Bacillus species. The inhibitory activity against C. lunata in dual culture assays strain SR/B-16 can be used for the formulation of bioactive (Alfonso and Villa 2002; Basha and Ulaganathan 2002; compounds for the treatment of C. gudauskasii diseases on Tendulkar et al. 2007). sugarcane seedlings. Bacillus and its relatives are attractive candidates for bio-controlling plant pathogens because they produce . . . Keywords Bacillus subtilis Culture filtrate Antifungal extracellular metabolites such as antibiotics lipopeptides . . Curvularia gudauskasii Hyphal abnormality Biocontrol and hydrolytic enzymes, which play an important role in the direct antagonism of plant disease fungi (Kildea et al. 2008;Yangetal. 2008; Saidi et al. 2009). Their Introduction interaction with filamentous fungi induces hyphae mal- formations related to their biological control mechanisms. Numerous plant species are affected by Curvularia plant The predominant morphological alterations are swelling pathogen fungi, mainly under climatic stress periods with and bulbous formation, hyphae distortion, cell wall high temperatures and environmental humidity. Curvularia darkness, cytoplasm aggregation, inhibition of spore ger- phytopathogen species produce spots in grains and seeds, as mination and hyphae tip rupture (Rahman et al. 2007; well as damage to plant leaves (Barrios and Pérez 2005). Prapagdee et al. 2008). In a previous study, five Bacillus subtilis rhizosphere : : : T. Orberá Ratón (*) Z. G. Giro M. Serrat Díaz S. R. Pérez strains exhibited a marked inhibitory effect on the plant Centro de Estudios de Biotecnología Industrial, pathogen fungi Curvularia lunata, Fusarium oxysporum Facultad de Ciencias Naturales, Universidad de Oriente, and Colletotrichum sp. (Orberá et al. 2009). This research Avenida Patricio Lumumba S/N, was aimed at evaluating if the bioactive compounds 90500, Santiago de Cuba, Cuba secreted by those B. subtilis antagonistic isolates are able e-mail: torbera@cebi.uo.edu.cu 546 Ann Microbiol (2012) 62:545–551 to generate morphological alterations related to the in vitro growth inhibition percentage (RGIP) based on the following growth inhibition of a Curvularia gudauskasii strain formula: causing damage to sugarcane seedlings. R1  R2 RGIP ¼  100% ð1Þ R1 Materials and methods where, Microorganisms R1 Radial growth of C. gudauskasii in the control plate R2 Radial growth of C. gudauskasii interacting with the Bacteria antagonistic B. subtilis strain Five aerobic endospore-forming bacterial isolates with antagonistic properties against plant pathogenic fungi were used (Orberá et al. 2009). The strains were previously Hyphal morphology identified as Bacillus subtilis and the 16S rRNA gene partial sequences accession numbers, deposited at the Following positive antibiosis test results, hyphal strands National Centre for Biotechnology Information Database from the border of the fungal colony nearest to the growth are: SR/A-1 [HQ025915], SR/B-8 [HQ025916], SR/B-16 inhibition zone were removed smoothly using a sterile [HQ025917], SR/B-17 [HQ025918], and SR/A-17 needle and placed on a microscope slides with lactophenol [HQ025919] (NCBI, http://www.ncbi.nlm.nih.gov). 28.5% (BDH Chemicals, Poole, UK) and examined under a light microscope (40×), looking for hyphal abnormalities. Fungi Microphotographs were taken using a digital camera (Canon Power Shot A640; Rahman et al. 2007). A phytopathogen strain of Curvularia gudauskasii was provided by the Phytopathology Laboratory strain collec- Bacterial growth kinetics tion at the National Sugarcane Research Institute (INICA), Havana, Cuba. fresh bacterial culture of B. subtilis SR/B-16 from a NA The microorganisms were maintained at −20°C on glycerol, slant previously grown for 18 h at 30°C, was inoculated at the Industrial Biotechnology Study Centre Culture Collec- into a 250 mL Erlenmeyer flask containing NB medium. tion (CCEBI) [http://www.aam.org.ar/cultivos_microbianos. The culture was incubated at 30°C for 12 h and shaken shtml]. Bacteria were subcultivated on nutrient agar vigorously at 150 rpm. For growth curve estimation, 1 mL (NA, Biolife Italiana, Milan, Italy) and nutrient broth from the pre-inoculum was inoculated into a 500-mL (NB, Biolife Italiana). Fungi were grown on potato Erlenmeyer flask containing 100 mL NB. The culture was dextrose agar (PDA, UNI–CHEM Chemical Reagents; then incubated for 72 h at 30°C. Cellular growth was http://www.unichem-chemicals.com/). determined spectrophotometrically by measuring changes in optical density (OD) with time at 620 nm. Bacillus subtilis strains with antifungal activity against Curvularia gudauskasii Characterization of bacterial culture filtrates The dual culture assay was performed in Petri dishes Bacterial culture was transferred to NB and incubated for containing PDA medium. A 5-mm diameter mycelial 6 and 21 h, while shaking vigorously at 150 rpm at 34°C. plug taken from a 5-day-old culture of C. gudauskasii Cells were removed by centrifugation at 5,000 rpm at 10°C was transferred to the center of the Petri dish. One for 10 min. The supernatant collected was filtered through a milliliter of each Bacillus subtilis strain suspension 0.2 μm pore size Millipore membrane. The protein and 6 −1 (10 CFU mL ) was inoculated at an equidistant point carbohydrate content, as well as the qualitative presence of (20 mm) from the Petri dish center, which was inoculated lipids were determined on the culture filtrates. Protein with the mycelial plug. The plates were incubated in the content was determined following the Lowry technique, dark at 30°C. Trials were done in triplicate and a control using a solution of bovine serum albumin (BSA; BDH, was prepared with the fungal culture without bacterial Poole, UK) as standard (Lowry et al. 1951). The absor- inoculation. The appearance of a growth inhibition zone of bance was read at 650 nm. Soluble carbohydrates were the fungal colony was taken as a positive result (Muhammad determined using the phenol–sulfuric method (Dubois et al. and Amusa 2003). The inhibitory effect was assessed 1956). Spectrophotometer readings were taken at 490 nm. −1 following Ezziyani et al. (2004), by calculating the radial A glucose solution (UNICHEM, 1.00 g L ) was used as Ann Microbiol (2012) 62:545–551 547 standard. The qualitative presence of lipids was determined using the Sudan III coloring technique (Díaz et al. 1989). A test tube with sterile medium was used as a negative control. Concentration of free-cell extracts Sterile extracts were separated with chloroform: methanol mixture (2:1) according to the Folch method (Folch et al. 1957). The organic extract was concentrated in vacuo, yielding a brownish gummy residue, which was suspended in 5 mL phosphate buffer at pH 6.5. The soluble phase was Fig. 1 In vitro radial growth percentage inhibition (RGPI) of kept at 4°C to be used later. Curvularia gudauskasii by Bacillus subtilis isolates. Different lower case letters indicate statistical differences at P≤0.005 Antagonism test with free-cell extracts Antagonism tests with free-cell extracts were carried out using Curvularia gudauskasii mycelium alterations the plate diffusion technique described by Vignolo et al. upon interaction with Bacillus subtilis antagonistic strains (1993). Sterile filtrates collected at 6 and 21 h of incubation were evaluated, as well as the organic and inorganic fractions Dual culture on Petri dishes of B. subtilis strains SR/A-1, obtained from the 21-h extracts. Aliquots of 100 μLof the SR/B-8 and SR/B-16 with C. gudauskasii is shown in crude extract were deposited in sumps opened in the surface Fig. 2. A growth inhibition zone produced by bacteria of the solid medium surface on Petri dishes, previously extracellular metabolites was observed around the fungal inoculated with a mycelium plug. The plates were incubated mycelium. This zone was visible next to isolates SR/A-1, at 30°C for 7 days until the appearance of mycelium growth SR/B-17 and SR/A-1 from the 3rd to the 5th incubation inhibition zones. Hyphal abnormalities were studied as day, when the fungal growth restarted; however, the rate described (Rahman et al. 2007). was lower and a loss of the typical dark color of mature Curvularia colonies was observed (Fig. 2d). As Curvularia Statistical analysis pigmentation is attributed to the presence of asexual conidia, this loss of color indicated that antagonistic B. subtilis strains inhibited spore formation in this plant The means and standard deviations of the inhibition of radial growth were calculated. Data were analyzed by one- pathogen fungus. No mycelial growth was seen around way analysis of variance (ANOVA). Significant differences strains SR/B-8 and SR/B-16 after 15 incubation days. (P≤0.05) among the means were determined by Duncan’s Necrosis and darkening colour were also observed in the multiple range tests, using the software Statgraphics Plus 5.1 border of the colony nearest these isolates (Fig. 2d). B. for Windows (http://www.statgraphics.com/statgraphics_plus. subtilis SR/B-16 showed the strongest antifungal effects, htm). and the study was continued using only this strain and its extracellular metabolites. Microphotographs showed hyphal swelling, bulb formation, a granular cytoplasm with an Results intense vacuolization, and absence of conidia (Fig. 2e–f). In vitro growth inhibition of Curvularia gudauskasii Cell-free extract effects of SR/B-16 on Curvularia by Bacillus subtilis gudauskasii morphology Growth inhibition of C. gudauskasii by B. subtilis isolates The encounter of C. gudauskasii growing mycelium and is represented in Fig. 1. Bacteria showed values of RGIP of SR/B-16 sterile filtrates collected during its active growth, between 41 and 71%, corresponding to the isolates SR/A- did not show any alterations on hyphae and colony 17 and SR/B-16, respectively. Strains SR/B-8 and SR/B-16 morphology. However, fungal cultivation with the extracts had similar significantly higher inhibitory effects; however, obtained at stationary phase (21 hours) corroborated that the latter was the most efficient. All the strains were they contain the metabolites implicated in bacterial anti- classified as moderately toxic according to the scale used fungal activity (Fig. 3). The in vitro interaction of C. by Ros et al. (2008), which considered as toxic only those gudauskasii with the collected sterile extract of stationary isolates inhibiting fungal growth by 100%. phase B. subtilis SR/B-16 generated a mycelium growth 548 Ann Microbiol (2012) 62:545–551 Fig. 2 In vitro interaction effects of B. subtilis antagonistic strains and vacuolization, bulb formation, swelling, no conidia); g fungal colony C. gudauskasii (40× magnification). a Control plate; b,c control in interaction with SR/B-16 sterile extracts; h C. gudauskasii hyphae mycelium; d dual culture of C. gudauskasii and B. subtilis isolates; e,f interaction with SR/B-16 sterile extracts (arrows swelling, vacuoliza- C. gudauskasii hyphae encounter with B. subtilis SR/B-16 (arrows tion, no conidia). IZ Growth inhibition zone, CE cell free extracts inhibition zone (Fig. 2g). The corresponding hyphae showing that they were consumed as nutrients. The microphotographs showed swelling, intense vacuolization qualitative presence of lipids was determined only in and no conidia formation (Fig. 2h). extracts collected at stationary phase. Characteristics of SR/B-16 free-cell extracts Antifungal effects of concentrated extracts of SR/B-16 Protein and carbohydrate contents in the sterile extracts The cell-free extract of SR/B-16 strain was partitioned into collected at 6 and 21 incubation hours were determined aqueous and organic phases in order to evaluate their effects (Fig. 3). Total protein content was higher in exponential on fungal morphology. The in vitro interaction of the phase, and decreased when active growth ended. Carbohy- organic fraction of B. subtilis SR/B-16 sterile extracts with drates decreased along with bacterial growth kinetics, C. gudauskasii caused abnormal appearance of the fungal Ann Microbiol (2012) 62:545–551 549 lycopersici isolated from tomatoes cultivars by more than 50% (Saidi et al. 2009). In order to determine how rhizobacterial isolates affect fungal growth, the presence of hyphal malformations in C. gudauskasii colonies was investigated using a dual culture assay with B. subtilis SR/B-16. The bacterial strain and its sterile crude extracts showed the highest antagonistic activity. The swelling and the absence of conidia observed in the plant pathogen fungus under the light microscope are abnormalities caused by antagonistic bacteria and conven- tional antifungal compounds interacting with filamentous fungi. Swelling has been detected in the presence of Fig. 3 Bacillus subtilis SR/B-16 growth on nutrient broth (NB). quitinases enzymes and antibiotics such as iturins, sub- Protein and carbohydrate concentrations in the crude extracts during tilisins and surfactins (Tendulkar et al. 2007; Rahman et al. growth stages are indicated 2007; Prapagdee et al. 2008). Swelling is also related to the cells. Microphotographs (Fig. 4) show cell wall thickening, disorganization of cellular actin filaments produced by a reduction in hyphae diameter and the absence of asexual secondary metabolites from antagonistic rhizobacteriae conidia. The interaction of C. gudauskasii with the (Deora et al. 2010). The absence of conidia has been inorganic fraction did not generate mycelium alterations. reported as one of the malformations generated by antifungal isolates of Paenibacillus lentimorbus, Burkhol- deria cepacia and Streptomyces hygroscopicus and their Discussion sterile filtrates (Chen et al. 2003; Rahman et al. 2007; Prapagdee et al. 2008). Both swelling and the absence of The present study explored, for the first time, the potential conidia were observed on Neurospora crassa grown in the use of Bacillus subtilis rhizosphere isolates for the presence of conventional chemical fungicides (Pereira and biological control of the sugarcane seedling pathogenic Said 2009). fungus Curvularia gudauskasii. In vitro fungal growth The necrosis observed in the mycelium border at the inhibition values coincided with previous results shown by interaction zone with the strains SR/B-8 and SR/B-16 is other researchers. In Cuba, inhibition of Curvularia probably related to fungal growth inhibition. It is well known phytopathogenic species by B. subtilis ATCC 6633 with that alterations in the apex of hyphae of filamentous organisms radial growth inhibition has been reported to be between 66 affect major biological processes such as growth, reproduc- and 100% (Castellanos et al. 2002). Rahman et al. (2007) tion, morphogenesis, nutrient absorption and protein secretion isolated Pseudomonas aeruginosa and Burkholderia cepa- taking place in the tips (Pereira and Said 2009). cia strains that strongly inhibited the growth of Colleto- The morphological alterations in C. gudauskasii seen upon trichum gloeosporoides by an average of 74 and 68%, application of crude extracts of SR/B-16 are less severe than respectively. Numerous Bacillus species inhibited the in those seen in dual culture assay with bacterial colonies. This vitro growth of a Fusarium oxysporum f.sp. radicis- suggests that bacteria act through a combination of at least Fig. 4 Morphological abnormalities of C. gudauskasii mycelium in encounter with organic extract (arrows distortion, hialinization, bulbs interaction with the organic concentrate of B. subtilis SR/B-16 crude formation, absence of conidia) extract (40× magnification). a Control mycelium; b,c mycelium 550 Ann Microbiol (2012) 62:545–551 two biological control mechanisms: nutrient competition and iturins, surfactins, and fengicins, excreted as secondary antifungal metabolite secretion. A similar phenomenon was metabolites. All play different roles in the growth and produced by a B. subtilis strain with inhibitory effects on survival of Bacillus species in their natural habitat, Trichoderma species contaminating commercial mushroom rhizosphere colonization and their biological control efficacy production (Chittihunsa et al. 2007). In vitro dual culture (Arguelles-Arias et al. 2009). However, to confirm our betweenanantagonistic Bacillus lentimorbus isolate and suggestions regarding the nature of the antifungal com- Colletotrichum gloeosporoides showed that nutrient compe- pounds from B. subtilis SR/B-16, more studies, including tition at early stages and antibiosis at advanced stages are characterization and purification tests, mass spectra and responses to their stress interaction (Lee et al. 2005). This NMR, will be needed. result is invaluable during the selection of an efficient biological control agent (BCA). Antifungal metabolite secretion is known to offer more advantages than competi- Conclusions tion, but the synergistic effects in a BCA strain increase considerably its effectiveness as a plant disease treatment. B. subtilis rhizosphere isolates were evaluated for their Bacillus subtilis SR/B-16 developed diauxic growth in NB potential for biological control of the phytopathogen C. culture medium, which may be attributed to the initial gudauskasii. The wild type strain SR/B-16 inhibits the in hydrolysis of peptone, a simple carbon source present in the vitro growth of the plant pathogen fungus by more than culture medium. After that, SR/B-16 entered a second lag 70%, probably through a combination of two biological phase, probably developing physiological mechanisms to control mechanisms: nutrient competition and the excretion hydrolyze beef extract, a more complex carbon source present of antifungal lipopeptides as secondary metabolites. Sterile in NB, showing the wider potential of this strain to grow in a filtrate collected at stationary phase contains the bioactive variety of carbon sources. Both peptone and beef extract compounds responsible for the alterations in morphology supplied B. subtilis SR/B-16 with the nutrients required for and growth pattern, as well as the absence of conidia, which growth. In the technical sheet of NB culture medium from are related to fungal growth inhibition. Therefore, we Biolife Italiana (http://www.biolifeit.com/biolife/upload/file/ suggest that B. subtilis SR/B-16 may be used as an Schede/TS-401810.pdf), the culture medium is referred to as inoculant for the biological control of diseases caused by beef peptone media. Peptone hydrolysis by Bacillus strains C. gudauskasii in sugarcane seedbeds. has been reported by Vasileva-Tonkova et al. (2007). Acknowledgment The authors are grateful for the assistance of Juan The presence of proteins in the sterile extracts of SR/B- Alfonso Ayala Serrano, PhD from the Molecular Biology Institute 16 is attributed to the ability of Bacillus and its relatives to “Severo Ochoa”, with the molecular identification of the Bacillus excrete numerous enzymes, signals and antifungal peptides isolates used in this research. (Arguelles-Arias et al. 2009). The decrease in the final protein content may be provoked by the production of References bacterial extracellular proteases. Razo et al. (2008) reported maximum levels of proteases in a B. subtilis strain at the end of its active growth. Alfonso I, Villa P (2002) Evaluación de la cepa de Pseudomonas spp. The detection of proteins and lipids in SR/B-16 crude cepa PSS en el control de hongos fitopatógenos de las semillas agámicas y botánicas de la caña de azúcar. 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Journal

Annals of Microbiology – Springer Journals

Published: Jun 30, 2011

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