Crop Protection 35 (2012) 29e35
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Screening and partial characterization of Bacillus with potential applications in biocontrol of cucumber Fusarium wilt Lihua Li a, Jincai Ma b, Yan Li a, Zhiyu Wang c, Tantan Gao a, Qi Wang a, * a
The MOA Key Laboratory of Plant Pathology, Department of Plant Pathology, College of Agronomy and Biotechnology, China Agricultural University, Beijing 100193, China Department of Environmental Sciences, University of California, Riverside, CA 92521, USA c Department of Environmental Science and Engineering, Tsinghua University, Beijing 100084, China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 16 March 2011 Received in revised form 25 November 2011 Accepted 12 December 2011
Bacullus strains are effective biocontrol agents of the cucumber wilt caused by Fusarium oxysporum f. sp. Cucumerinum. In this study, a total of 400 Bacillus samples were isolated from surface-sterilized roots of cucumber plants grown in greenhouses and ﬁelds, and were screened using a modiﬁed gnotobiotic system for their capability in controlling Fusarium wilt of cucumber. A strain designated as B068150 showed a high potential in control of the Fusarium wilt, with biocontrol effectiveness up to 50.68% in seedling stage in greenhouse experiments. Interestingly, B068150 showed no obvious antagonistic activity to F. oxysporum f. sp. Cucumerinum on potato dextrose agar plate. B068150 was identiﬁed as Bacillus subtilis by using morphological, physiological, biochemical tests, cellular fatty acids analysis, and Biolog-based substrate utilization test. In addition, 16S rRNA gene and gyrA gene-based phylogenetic analysis illustrated that B068150 exhibits high levels of similarity to known Bacillus species. Therefore B068150 was ﬁnally designated as B. subtilis B068150. The pot experiments results in greenhouse indicated that B. subtilis B068150 could be a promising agent in biocontrol of Fusarium wilt of cucumber, which might help to minimize the yield loss of cucumber caused by F. oxysporum f. sp. cucumerinum in north China. Ó 2011 Elsevier Ltd. All rights reserved.
Keywords: Cucumber Fusarium oxysporum f. sp. cucumerinum Bacillus subtilis Gnotobiotic system Biocontrol
1. Introduction Fusarium wilt of cucumber is a severe soilborne fungal disease caused by Fusarium oxysporum f. sp. cucumerinum. Typical symptoms of wilt disease in cucumber plants include necrotic lesions, vascular wilt, and roots wilts, which ultimately lead to severe yield loss (Vakalounakis et al., 2004). The disease generally manifested in young and mature plants throughout all of the cucumber-growing stages (Ahn et al., 1997). Fusarium wilt causes more damage when the vigor of cucumber plants is signiﬁcantly reduced under unfavorable microclimatic conditions (Tok and Kurt, 2010). Fusarium wilt of cucumber was widely found in China and it is also a worldwide issue in cucumber-growing areas. Cases on Fusarium wilt of cucumber have been well documented in Crete, Greece, North and south American countries, Israel, Japan, France, Spain, Korea, and other regions (Vakalounakis et al., 2004; Pavloua and Vakalounakis, 2005). In the past two decades, the most popular method to control soilborne plant pathogens was soil fumigation with methyl bromide or other fumigants (Pavloua and Vakalounakis, 2005). Both chemical and biological methods have been applied to control Fusarium wilt of * Corresponding author. Tel.: þ86 10 627 314 60; fax: þ86 10 628 104 32. E-mail address: [email protected]
(Q. Wang). 0261-2194/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.cropro.2011.12.004
cucumber. Since most of the fumigants, e.g. methyl bromide, are ozone depleting chemicals, the use of those fumigants has been banned under the Montreal protocol by the end of 2004 (Gullino et al., 2003; Minuto et al., 2006). In contrast, the biological control as an alternative approach to control soilborne plant diseases has attracted worldwide attention. Bacillus spp. was shown to be a promising biocontrol agent to control Fusarium wilt of cucumber. For example, B. subtilis ME488 and B. subtilis D1/2 have been formulated as biofertilizers to effectively control Fusarium wilt disease of cucumber (Chung et al., 2008). Similar Bacillus strains have been isolated from cucumber-growing ﬁelds in China, such as B. subtilis SQR-5 and B. subtilis B579 which have a great potential in biocontrol of Fusarium wilt of cucumber (Zhang et al., 2008; Chen et al., 2010). In combination with another biocontrol strain Paenibacillus polymyxa SQR-21, B. subtilis SQR-5 was found to be a super biofertilizer which could control F. oxysporum wilt disease of cucumber effectively. B. subtilis B579 exhibited a good biological control effect by seed-soaking. Bacillus strains have the capacity to form spores which survive and remain metabolically active under harsh environmental conditions (Rodgers, 1989), and these properties make them ideal biocontrol agents. The choice of screening methods is critical for successful identiﬁcation of efﬁcient biocontrol agents against soilborne pathogens.
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Ideally, the candidate biocontrol organism could be successfully screened on whole plants rather than in cell culture or on leaf disks in vitro (Weller, 1988). However, the large-scale screening work on whole plants is generally time-consuming and labor-intensive, thus the traditional screening method, dual culture assay, is very popular (Oldenburg et al., 1996). In this method, antagonisticactivity-positive biocontrol agents form clear inhibition zones on potato dextrose agar (PDA), malt extract agar, potato dextrose broth or other mediums (Swadling and Jeffries, 1996), while antagonisticactivity-negative biological agents might not be successfully identiﬁed during screening processes. It is a matter of fact that most of the well documented Bacillus biocontrol agents are strongly antagonistic to plant pathogens (Chung et al., 2008; Zhang et al., 2008; Chen et al., 2010). Reports on Bacillus strains with less antagonistic activity are scarce, and alternative screening strategy is required to identify the potential biocontrol strains with decreased antagonistic activity toward plant pathogens. The gnotobiotic system has been used for testing plants for disease control in some studies (Timmusk et al., 2005; Haggag and Timmusk, 2007). This system might be ideal for biological control agents screening, especially in screening of biocontrol agents that have relatively weak antagonistic activity toward fungal pathogens. Our objective was to screen biological control agents that might effectively control Fusarium wilt disease of cucumber at the seedling stage by using a gnotobiotic system in vivo. 2. Materials and methods 2.1. Isolation of Bacillus from cucumber root tissues Cucumber plants from eight ﬁelds in major cucumber-growing regions of China including Beijing, Hebei, Xinjiang, Ningxia and Neimenggu were sampled over different growing seasons in 2007 and 2008, and both healthy and diseased plants were included. The isolation of Bacillus spp. was performed according to the method reported previously (Cavaglieri et al., 2004). Roots tissues of cucumber plants were washed with sterile distilled water and the surface was sterilized by immersing them in 3% NaOCl for 10 min, and then soaked in 70% ethanol for 1 min for further sterilization, ﬁnally rinsed ﬁve times with sterile distilled water. The sterility of the water used for the last rinse of roots was checked by inoculating an aliquot (50 ml) on nutrient agar (NA) plate and subsequently examining the presence/absence of colonies formed on the plates. Only surface-sterilized roots were used to isolate Bacillus. Roots were then cut into 1 mm-long pieces and placed in a sterile mortar and mashed with sterile water. The resultant suspension was pasteurized for 10 min at 80 C, and then spread out on NA plates after ten-fold serial dilution. The plates were then incubated at 30 C for 48 h. The colonies formed on NA plates were transferred onto LB agar plates for further characterization and identiﬁcation. All isolates were maintained on NA plates at room temperature or stored in nutrient broth containing 15% glycerol at 80 C. 2.2. Screening of biocontrol agents for Fusarium wilt of cucumber Cucumber seeds of susceptible cultivar Changchun Mici, were surface-sterilized by soaking in 3% NaOCl solution for 10 min, followed by ﬁve times washing with sterile distilled water. Then they were placed on plates (9 cm in diameter) covered with sterile wet ﬁlter papers, and incubated at 30 C for 24 h for germination. More than 30 germinated seeds were soaked in 5 ml suspension (1 108 Colony Forming Unit ml1) of Bacillus spores (one of Bacillus strains isolated in this study) for 1 h. For the control experiments, the same number of germinated seeds was soaked in sterile distilled water instead of the Bacillus spores suspension. The treated germinated
seeds were then transferred onto wet ﬁlter papers in new dishes to allow Bacillus strains to colonize on germinated seeds. All dishes were incubated at 30 C for 24 h until the embryo grew to 1.0e1.5 cm in length. Pathogenicity test was performed using a root-dipped method (Johnston, 1968). The pathogen (F. oxysporum f. sp. cucumerinum) cultured on PDA plates was transferred to a 100 ml ﬂask containing 50 ml of PDA liquid medium. The ﬂask was shaken at 160 rpm at 26 C. Six days later, the culture was ﬁltered with a two-layer sterile pledget to collect spores, and the spores were washed for three times with sterile water. The concentration of the pathogen in conidial suspension was adjusted to 1 106 CFU ml1. Over 24 cucumber embryos measuring 1.0e1.5 cm in length were subsequently inoculated with the pathogen by dipping embryo root into the pathogen conidial suspension. Then eight embryos were sown in one culture ﬂask containing minimal salt (MS) agar medium, and triplicate ﬂasks were prepared. Subsequently the germinations were grown for one week in a growth chamber (16 h light at 26 C and 8 h dark at 20 C). Three treatments were made, including: 1) Root-dipping germinations inoculated with B068150 in pathogen conidial suspension (1 106 CFU ml1); 2) Root-dipping germinations not inoculated with B068150 in pathogen conidial suspension (1 106 CFU ml1); and 3) Root-dipping germinations not inoculated with B068150 in sterile distilled water (no pathogen). All of the operations were conducted under sterile conditions. The MS medium containing the macronutrients, micronutrients, and vitamins was prepared following the recipe reported previously (Murashige and Skoog, 1962). The pH of MS medium was adjusted to 6.2, and agar was added to a ﬁnal concentration of 0.45%. Symptoms of wilt disease on cucumber plants were generally observed after the plants grew for seven days in a growth chamber. The least and the most devastatingly diseased seedlings were removed, and the disease severity index (DSI) was assessed with a 0e4 visual scale (Chen et al., 2010) with modiﬁcation. The DSI was based on the percentage of roots discolored 0, no symptom; 1, roots discolored from 0 to 25% and plants showed slight dwarﬁng; 2, roots discolored from 25% to 50% and leaves discolored between major veins; 3, root discolored from 50% to 75% and caudexes dehisced longitudinally; and 4, roots discolored from 75% to 100% or plants died. Pathogenicity tests were conducted in triplicate for each treatment. The biocontrol effect was assessed according to the method of Chen et al. (2010). The Bacillus isolate showed best biocontrol effect was further conﬁrmed using the same method as described above. 2.3. Pot experiment The pot experiment was conducted in a greenhouse located at China Agriculture University, China, during April to May in 2008. The temperature ranged between 24 and 28 C during day and 15e20 C at night. The relative humidity was maintained between 60% and 80%. Sterilized seeds were used in the pot experiments. The pot experimental design included 4 treatments: 1) Soaking the germinations treated with B068150 in pathogen conidial suspense (5 106 CFU ml1) for 0.5 h; 2) Soaking the germinations treated with carbendazim (100) (Omar et al., 2006) in pathogen conidial suspension (5 106 CFU ml1) for 0.5 h without being inoculated with B068150; 3) Soaking the germinations treated without B068150 and carbendazim in pathogen conidial suspension (5 106 CFU ml1) for 0.5 h; and 4) Soaking the germinations treated without B068150, pathogen, and carbendazim in sterile distilled water. The germinated seeds were then transferred to sterile soil, with ﬁve seeds in one pot in triplicate. The DSI and biocontrol effect were assessed after the plant were grown for 25 days in greenhouse until infected plants exceeded 50% of total plants.
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2.4. In vitro test of antagonistic activity between strain B068150 and F. oxysporum f. sp. cucumerinum Isolate B068150 was tested for antagonism in vitro against the phytopathogenic F. oxysporum f. sp. cucumerinum by using a direct dual culture method (Oldenburg et al., 1996). The pathogenic fungus cake (0.5 cm in diameter) was transferred to the centre of PDA plate (9 cm in diameter), and B068150 was inoculated by dripping 1 ml B068150 suspension (1 108 CFU ml1) to the periphery of the agar surface. The PDA plates were incubated at 26 C for seven days, and then antagonism effect was assessed by checking the size of the inhibition zone formed on the PDA plates. Two agar concentrations (10% and 15%) were used to make PDA plates. The antagonism test was conducted in triplicate. 2.5. Characterization of strain B068150 The morphological and physiological properties of strain B068150, including cell shape, motility, formation of spores and Gram stain, were characterized. The growth of B068150 was assayed on various substrates including citrate and starch. The physiological properties, such as methyl red test, catalase activity, VogeseProskauer test, anaerobic growth, pH range of growth, range of growth temperature and tolerance to NaCl, were conducted according to protocols described previously (Roberts et al., 1994). All assays were conducted at 30 C, except for the temperature test. Whole-cell fatty acids analysis was performed by using the Sherlock Microbial Identiﬁcation System (MIS) TSBA6.0 following standard procedures (Microbial ID, Newark, DE, USA). In brief, strain ;B068150 was grown on TSBA medium for one day at 30 C. The whole-cell fatty acids were extracted following the protocol supplied by MIS and the composition of the fatty acids was determined by using a gas chromatograph (model 6850, Agilent) equipped with a capillary column (Agilent,19091B-102E, 25 m 200 mm 0.33 mm). The analysis was conducted with the following procedure: injection at 170 C; ramping at a rate of 5 C min1 to a ﬁnal temperature of 260 C which was maintained for 18 min; then ramping at a rate of 40 C min1 to a ﬁnal temperature of 310 C which was maintained for 20.75 min. N2 was used as carrier gas with a constant ﬂow rate of 0.469 ml min1. Numerical analysis and bacterial identiﬁcation were done with standard MIS Library Generation Software (Microbial ID, Newark, DE, USA). Strain B068150 was tested for their carbon sources utilization pattern using BIOLOG GEN III system (Omnilog, Hayward, CA) according to the manufactures’ protocols (Biolog, CA, USA). The MicroPlates were read automatically by BIOLOG GEN III system and the supplied database was used to characterize B068150. 2.6. Phylogenetic identiﬁcation of B068150 based on 16S rRNA and gyrA gene sequence analysis A universal primer set consisting of 63F and 1378R, which is speciﬁc for bacteria 16S rDNA, was used to amplify the 16S rDNA gene (Knapp et al., 2009). The PCR ampliﬁcation was performed by using a Taq DNA polymerase kit (Beijing TransGen Biotech Co., Ltd) with the following thermocycler protocol which included an initial denaturation at 95 C for 3 min followed by 30 cycles of denaturation at 95 C for 1 min, annealing at 55 C for 30 s, and extension at 72 C for 2 min; and a ﬁnal extension at 72 C for 10 min. According to a protocol described previously (Roberts et al., 1994), the primer set consisting of p-gyrA-f and p-gyrA-r was used to amplify the gyrA gene with the following thermocycler protocol which included an initial denaturation at 95 C for 3 min followed by 30 cycles of denaturation at 95 C for 1 min, annealing
at 60 C for 30 s, and extension at 72 C for 1 min, and a ﬁnal extension at 72 C for 10 min. PCR products with expected sizes (1.37 kb for 16S rDNA and 1.025 kb for gyrA gene) were ligated into pMD19-T vector (Takara Co. Ltd.) and the recombinant plasmid was harvested from an overnight LB culture using StarPrep Plasmid Miniprep Kit (GenStar Biosolutions Co. Ltd) and sent to Sanboyuanzhi Biotechnologies Co. Ltd. for sequencing. Phylogenetic tree of strain B068150 based on gyrA gene was constructed using the neighbor-joining method of Mega 4.1 software (Tamura et al., 2007). 2.7. PCR detection of antibiotic biosynthesis genes PCR was used to amply three genes (fenB, srfA and sfp), which are reported to involve in fengycin and surfactin biosynthesis in Bacillus spp, using primer sets previously described (Chung et al., 2008). The PCR ampliﬁcation was performed by using a Taq DNA polymerase kit (Beijing TransGen Biotech Co., Ltd). Thermocycler protocol included an initial denaturation at 95 C for 3 min followed by 30 cycles of denaturation at 95 C for 1 min, annealing at 55 C for 30 s, and extension at 72 C for 1 min, and a ﬁnal extension at 72 C for 10 min. PCR products were ligated into pMD19-T vector and sequenced by Sanboyuanzhi Biotechnologies Co. Ltd. 3. Results 3.1. Screening of isolates with a modiﬁed gnotobiotic system Four hundred Bacillus strains were isolated from the surfacesterilized root tissues of cucumber plants according to the isolation procedures described above. All of the isolates were screened with a gnotobiotic system. Isolate B068150 showed distinctive inhibition to cucumber wilt disease out of all isolates after seven days of growth in a growth chamber. The results of disease control tests were illustrated in Table 1 and the symptoms of Fusarium wilt on cucumber were shown in Fig. 1. It was observed in the gnotobiotic experiment that the DSI was reduced to 39.17% in Fusarium-inoculated plants treated with strain B068150 when compared to Fusarium-inoculated plants treated without B068150 (Table 1). Overall, strain B068150 could signiﬁcantly reduce the disease severity of the cucumber plants caused by F. oxysporum f. sp. cucumerinum (Fig. 1). When treated with the pathogen only, the growth of main roots of the cucumber plant was signiﬁcantly inhibited. Few and weak lateral roots were observed in the cucumber plants in disease control experiments. While after treated with B068150, the cucumber plant germinations sustained better growth of main roots and the roots were strong after seven days culture. Additionally, plants treated with B068150 showed better overall growth and roots coloring than those in the disease control. Thus, the strain B068150 showed the capability in protecting the main roots of cucumber plant against the pathogen infection.
Table 1 Biological control effect of strain B068150 on Fusarium wilt of cucumber in gnotobiotic system screening. Treatment
Disease severity indexa
Biocontrol effective (%)a
Seed-soaking with B068150 Pathogen control Untreated control
39.17b 56.67a e
30.88 e e
a Each of the values is the means of eight independent experiments. Lowercase letters indicate statistically signiﬁcant at P ¼ 0.05.
L. Li et al. / Crop Protection 35 (2012) 29e35 Table 2 Biological control effect of strain B068150 on Fusarium wilt of cucumber in pot experiment. Treatment
Disease severity index (%)a
Seed-soaking with B068150 Carbendazim control Pathogen control Untreated control
8.33ab 6.67b 16.67a e
50.68 60.78 e e
a Each of the values is the means of three experiments. The lowercase letters indicate statistically signiﬁcant at P ¼ 0.05.
Fig. 1. Symptoms of Fusarium wilt on cucumber cultivar “Changchun Mici” in gnotobiotic system. The numbers represents: 1-Disease control, 2-treating with B068150 and F. oxysporum f. sp. cucumerinum and 3-healthy control.
Application of strain B068150 in the germination seeds resulted in a lower Fusarium wilt disease incidence in pot experiment (Table 2). The biocontrol effect of strain B068150 was obvious (50.68%) and was comparable to that of the chemical control agent (carbendazim) treatment (60.78%) in the pot experiments. This further indicates the efﬁciency of strain B068150 in controlling cucumber wilt disease. 3.2. In vitro antagonism test The in vitro antagonism test on the plant soilborne pathogen F. oxysporum f. sp. cucumerinum and strain B068150 was carried out with the dual culture assay method on PDA plate. Generally, the fungus could grow on PDA agar just as well as strain B068150. The antagonisms test showed that the biocontrol strain B068150 exhibited slender strips of fungal growth inhibition on PDA containing 10% agar. But there was no clear antagonistic activity against F. oxysporum f. sp. cucumerinum on PDA containing 15% agar (Fig. 2). 3.3. Identiﬁcation of biocontrol strain B068150 The morphological, biochemical and physiological characteristics of strain B068150 were listed in Table 3. The biocontrol strain B068150 was found to be a Gram-positive, endospore-forming, motile, and catalase-positive rod bacterium. It was an anaerobenegative and was capable of utilizing citrate. In addition, preliminary taxonomic identiﬁcation of strain B068150 was carried out by performing a fatty acid analysis. It was classiﬁed as a B. subtilis strain with a similarity index greater than 0.3 in the TSBA6 6.10 Library and greater than 0.9 in the CLIN6 6.10 Library. 13-methyl tetradecanoic acid (iso-15:0), 12-methyl-
tetradecanoic acid (anteiso-15:0) and 15-methyl hexadecanoic acid (iso-17:0) were major whole-cell fatty acids. The result of BIOLOG GEN III System showed that the B068150 was likely B. subtilis because a high similarity index, 0.72, was observed at a decent probability level (71.9%). Based on 16S rDNA gene sequence analysis, it was found that the closest relatives of strain B068150 were B. subtilis subsp. subtilis JCM 10629 (99%) and Bacillus licheniformis strain B425 (99%). Strain B068150 was further characterized by sequencing gyrA gene to assess its taxonomical position (DNAMAN 5.0 and Mega 4.0). Its partial gyrA gene sequence was compared with gyrA sequences in some available Bacillus strains found in GenBank. Strain B068150 showed a high similarity with B. subtilis. It was found that the closest relatives were B. subtilis subsp. subtilis strain 168 (99%). Sequence similarity of strain B068150 with other B. subtilis validly published names was more than 94%. A phylogenetic tree was constructed at the basis of gyrA partial sequences, and Bacillus species were presented in Fig. 3. The results showed that strain B068150 stood close to B. subtilis. Based on the results of morphologic, physiological, biochemical characteristics and 16S rRNA, gyrA sequence analysis, Biolog system, as well as fatty acids test, strain B068150 was ﬁnally identiﬁed as B. subtilis and thus named as B. subtilis B068150. The partial 16S rRNA sequence and gyrA sequence of strain B068150 were submitted to the database of GenBank, and the submission number is respectively HQ589344 and HQ589343. 3.4. PCR of antibiotics produced by strain B068150 PCR products with expected sizes (671 bp for fenB gene and 676 bp for sfp gene fragments, respectively), further sequence analysis showed the resultant PCR products shared 99% identity to fenB and 99% identity to sfp, respectively. No amplicon with the expected size was obtained using a primer set targeting srfA, which is involved in surfactin biosynthesis. Sequence analysis of the sfp region also revealed four base substitutions and no base insertion within the open reading frame coding for sfp. 4. Discussion The stratagem of biocontrolling soilborne plant diseases has become an important approach for creating a long-lasting effect and facilitating sustainable agriculture. Some B. subtilis strains, which could biocontrol Fusarium wilt of cucumber, were initially screened with the dual culture method (Chung et al., 2008; Raza et al., 2009; Gajbhiye et al., 2010), in which the strains showed clear inhibition zone to pathogen on PDA plates. Then the candidates were tested in pot experiment to verify the ability to control disease. B. subtilis EM488 was screened for in vitro inhibition of Pythium ultimum, Rhizoctonia solani, F. oxysporum and Phytophthora capsici on PDA plates. Its biocontrol function was possibly attributed to the production of bacilysin and iturin (Chung et al., 2008). Similarly, B. subtilis B579, screened with the dual culture method, showed
L. Li et al. / Crop Protection 35 (2012) 29e35
Fig. 2. Strain B068150 (B) isolated from roots of cucumber showing no obvious antagonistic activity in dual culture study against fungal pathogens Fusarium oxysporum f. sp. cucumerinum (F) on PDA with 10% agar (2A) and on PDA with 15% agar (2B).
a good preventive effect on suppression of Fusarium wilt of cucumber in pot experiments (Zhang et al., 2008; Chen et al., 2010). The antagonistic effect of B. subtilis strains against F. oxysporum might be attributed to the production of chitinase, b-1, 3-glucanase, siderophores, indole-3-acetic acid (IAA), and hydrogen cyanide (HCN) (Chen et al., 2010). In our study, isolate B068150 was initially screened on plants in vivo by the modiﬁed gnotobiotic system, and then its biocontrol effect was tested by dual culture method. It was found that strain B068150 showed potential biocontrol effect to Fusarium wilt of cucumber, while not inhibiting the growth of
Table 3 Morphological and physiological characteristics of strain B068150. Characteristics
Cell shape Motility Form of spore Gram stain Utilization of citrate Catalase activity Hydrolysis of starch Methyl red test VogeseProskauer test Anaerobic growth Tolerance to NaCl Range of growth pH (Optimum) Temperature (Optimum)
Rod-shaped Positive Ellipsoidal Positive Positive Positive Positive (strong) Negative Positive Negative <14% 4.0e10.0, 5.0e9.0 15e50 C, 28e37 C
Fig. 3. Rooted neighbor-joining tree based on partial gyrA nucleotide sequences (900 bp). The percentage numbers at the nodes indicate the levels of bootstrap support based on neighbor-joining analyses of 1000 resampled data sets. The scale bar indicates 0.05 nucleotide substitutions per nucleotide position. The accession numbers of gyrA gene in the selected Bacillus strains were shown in brackets.
L. Li et al. / Crop Protection 35 (2012) 29e35
pathogen on PDA plates. The results revealed that some potential biocontrol strains that did less antagonistic activity toward the pathogen on PDA plates would be overlooked in the screening experiment by using the dual culture method. Most of the screening of potential biocontrol Bacillus was based on the antifungal activity (Zhang et al., 2008; Chen et al., 2010), those Bacillus spp. that do not have signiﬁcant antifungal effect will not be recognized as top biocontrol candidates for further characterization. The gnotobiotic system was widely used to study the interactions among plants, biocontrol microbes and pathogens (Trofymow et al., 1980; Simons et al., 1996). In this study, we screened Bacillus isolates from the roots of cucumber plants by using a modiﬁed gnotobiotic system. As the gnotobiotic screening system could avoid ﬁeld-soil variables, it should be a useful method. The result of pot experiments used to evaluate the modiﬁed gnotobiotic system was also in agreement with gnotobiotic studies. Our ﬁnding showed that strain B068150 is a potential biocontrol agent to control Fusarium wilt disease of cucumber. The taxonomy identiﬁcation of the screened biocontrol bacteria has increasingly come to rely on molecular biological methods. 16S rRNA sequencing is used widely as an alternative method to identify microbial organism, but it is not applicable for some Bacillus species with high pairwise 16S rRNA similarity values. Some genes like gyrA, gyrB, polC and rpoB have been proposed to provide conclusive evidence on the evolutionary relationships of B. subtilis and related taxa (Roberts et al., 1994; Wang et al., 2007). According to Jongsik and Kyung’s study (Chun and Bae, 2000), the inferred evolutionary relationships were based on direct sequence data of one gene, gyrA. All of the examined Bacillus strains showed almost identical 16S rRNA sequences, but signiﬁcantly low gyrA NT similarities (Jongsik and Kyung, 2000). The gyrA sequences provided sufﬁcient information to design species-speciﬁc nucleic acid probes and PCR primers for monitoring and screening industrially important B. subtilis-like isolates from natural sources. Likewise, we were unable to identify strain B068150 only with 16S rDNA sequence analysis. Therefore, we combined the gyrA sequence, morphological, physiological characteristics, cell fatty acids and Biolog analysis to identify B068150. Ultimately we identiﬁed B068150 as B. subtilis. In our experiments, isolate B068150 was able to control Fusarium wilt of cucumber at the seedling stage. It could also colonize strongly on the roots of cucumber and survive in natural soil (data not shown). Most of biocontrol agents are roots-colonized and antagonistic to the soilborne pathogen in the soilemicroorganismeplant system. The mechanisms of some biocontrol agents have been elucidated, including antibiosis (Koumoutsi et al., 2004; Cazorla et al., 2007; Kavroulakis et al., 2010), extracellular enzymes (Singh et al., 1999), induction of systemic resistance (Liu et al., 1995), competition for nutrients or for infection sites (Larkin and Fravel, 1999), and formation of bioﬁlm (Bais et al., 2004; Haggag and Timmusk, 2007). Some B. subtilis strains applied to seeds or seedlings have been found to be effective in suppressing soilborne diseases through inducing systemic resistance in the treated plants (Kloepper et al., 2004; Szczech and Shoda, 2006). The B. subtilis B068150 strain identiﬁed in this study could not form clear inhibition zones on PDA containing 15% agar, but could form slender inhibition strip on PDA containing 10% agar (Fig. 2), which implied that strain B068150 might produce a relatively small amount of antibiotics or enzymes to suppress the growth of the plant pathogen. B. subtilis is well known for producing antifungal compounds such as surfactin and fengycin (Stein, 2005). In the present study, some genes have been successfully ampliﬁed, including fenB involved in fengcin biosynthesis, and sfp involved in surfactin biosynthesis.The srfA is one of the regulation genes in surfactin biosynthesis (Coutte et al., 2010). However, PCR ampliﬁcation of srfA in strain B068150 was not successful. Sequence analysis of the sfp in strain B068150
revealed four base substitutions. Similarly, it was reported that the failure of strain B. subtilis 168 to produce surfactin is partly due to one base (adenine) insertion at position 634, and ﬁve base substitutions in sfp (Nakano et al., 1992). So strain B068150 could produce surfactin, but its activity might be low due to base substitutions. Our preliminary study showed that B068150 also had strong ability to form bioﬁlm (data not shown), which might be related to the biocontrol. Recent research mentioned that the bioﬁlm formed by B. subtilis just like a kind of biobarrier on the roots to protect plants from pathogens being infected (Bais et al., 2004; Morikawa et al., 2006). Obviously, additional work is needed to elucidate the mechanisms of the biocontrol effect of B. subtilis B068150. Acknowledgment This work was supported by grants from Beijing Natural Science Foundation (Grant number: 6101001) and National Natural Science Foundation of China (Grant number: 30871665). 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