Introduction
The pathogen
Botrytis cinerea Pers. causes serious losses in more than 200 crop species worldwide (
Williamson et al., 2007). Currently, the use of synthetic fungicides is the primary method to control post-harvest fungal decay (
Chen et al., 2008). However, there is a continuing need for more effective and safer fungicides, especially those with novel modes of action and resistance-breaking properties, and natural products have a key role to play in the search for such compounds. Scientists have focused on biological methods to protect crops from invasion and infection by the pathogen (
Liu et al., 2007). The management of pathogens by the use of antagonistic microorganisms or their secondary metabolites is now considered to be a feasible disease control technology (
Han et al., 2005).
B. cinerea is the most destructive on mature or senescent tissues of dicotyledonous hosts, but it usually penetrates into plant tissue at an earlier stage in crop development and remains quiescent for a considerable period before rapidly rotting tissues under a conducive environment, and the host physiology changes. Therefore, serious damage is caused following the harvest of apparently healthy crops and the subsequent transport to distant markets where the losses become evident (
Williamson et al., 2007). Endophytes, living for most of their life cycles inside healthy plant tissues, are mutualistic to their hosts. In particular, endophytic bacteria are thought to interact closely with their host plants and can therefore potentially be used as biological control agents in sustainable crop production (
Sturz et al., 2000). A variety of endophytes have been reported to have antagonistic activities toward bacterial and fungal pathogens (
Lodewyckx et al., 2002;
Sessitsch et al., 2004). At least seven products have been approved for use on food and non-food plants in greenhouses, under plastic tunnels, or in the fields in different countries. They have achieved niche markets in situations where heavy use of conventional fungicides is restricted because of accumulating residues or because of the restriction imposed by importing countries (
Elad and Stewart, 2004).
The aim of the present study was to isolate endophytic bacteria from Lycopersicumesculentum Mill. (tomato), Speranskiae tuberculatae (tuberculation speranskia herb), and Dictamnus dasycarpus Turcz. Densefruit pittany koot-bark which showed a strong inhibition on the growth of B. cinerea. A potential endophytic antagonist, Bacillus subtilis, denoted as EB-28, effective against the growth of B. cinerea, was identified in healthy stems of L. esculentum by in vitro and in vivo screening techniques and was characterized by morphology, physiological tests, and 16S rDNA sequence analysis.
Materials and methods
Isolation of endophytic bacteria
Healthy stems from L. esculentum and S. tuberculata, and stem bark from D. dasycarpus were collected from Baoding, Hebei, in April 2007. The endophytic bacteria were isolated according to the method described by
Liu et al. (2001). Three segments were randomly cut from each stem or bark. They were first washed in running water and then had their surface sterilized. The segments were washed again using sterile distilled water in autoclaved Petri dishes and then sectioned to 0.5 cm length using a small knife and placed on potato dextrose agar (PDA: potato, 200 g; dextrose, 20 g; agar, 15 g; distilled water, 1 L). After incubation for 1-2 days at 30°C in the dark, bacteria appeared on the plates and were individually isolated as single colonies on PDA.
Pathogen inoculum
A strain of B. cinerea (MYB01) used for laboratory trials had been isolated from infected tomato fruits with the typical symptom of gray mold in Baoding, China, and identified by its morphological characteristics. The fungal pathogen was cultivated on PDA plates for 10 days at 25°C until sporulation. A monospore isolate was maintained on PDA at 4°C and was subcultured onto fresh PDA plates at one-month intervals. Fresh, 5-mm-diameter mycelia plugs that were cut from the edge of a B. cinerea colony that was incubated for 5 days at 25°C were used as inoculum.
Tomato leaves and cucumber seedling cotyledons
Detached tomato leaves and cucumber seedling cotyledons were used for bioassay. The fourth leaves of the tomato plants (Lycopersicum esculentum cv. Hezuo908) and the eight-day-old cucumber seedling cotyledons (Cucumis sativus L. cv. Chuangchunmici) were uniform in size and free from wounds and lesions. Thereafter, the leaves were surface-sterilized by soaking in 2% aqueous sodium hypochlorite for 3 min. They were then thoroughly rinsed with sterile distilled water, dried by using sterile filter papers, and finally put into Petri dishes that contained 0.5% water agar medium (Jiao et al., 2009).
In vitro antagonism experiments
The inhibitory effects of endophytic strains on the growth of
B. cinerea were tested in Petri dishes containing the PDA medium. A 5-mm mycelium disc cut from a 5-day-old culture of
B. cinerea was deposited in the center of the plates, and bacteria from 2-day-old cultures were streaked across both sides of the mycelium disc approximately 2.5 cm away from the disc center. The plates were then incubated at 25°C for 4 days. The inhibition on fungal growth was evaluated by the reduction percentage of mycelium expansion compared to control plates without bacteria following the formula of
Whipps (1987): (
R1–
R2)/
R1×100, where
R1 is the farthest radial distance (measured in millimeters) grown by
B.
cinerea after 4 days of incubation in the direction of the antagonist (a control value), and
R2 is the distance of fungal growth from the point of inoculation to the colony margin in the direction of the antagonist. All
in vitro antagonism assays were made in triplicate (Fig. 1).
In vivo antagonism experiments
For each experiment, fresh cultures of the pathogen and the bacterial antagonists were used. To evaluate their antagonistic activities against B. cinerea on the detached tomato leaves, all bacterial strains selected from in vitro tests were grown for 48 h on Luria-Bertani (LB) medium at 30°C, shaken at 150 r•min-1. The cells were collected by centrifugation at 4000 r•min-1 and re-suspended with sterilized saline solution (1% NaCl). Bacterial suspension was adjusted to 1×108 colony-forming units (CFU)•mL-1. The bacterial concentration was determined by dilution plating on LB plate. After the bacterial suspension was sprayed on the surface of the detached tomato leaves, the leaves were allowed to air dry in a sterile cabinet for up to 60 min, followed by inoculation with a mycelium plug at the center of each leaf. The control treatments included bacterial candidate alone (non-Botrytis inoculated leaves, positive control) and distilled water (Botrytis-inoculated leaves, untreated control). The leaves were then stored in a growth chamber with 16 h of light and 8 h of darkness at 24°C for 48 h. The percentage of disease reduction of the gray mold on the tomato leaves was calculated using the following formula:
Reduction rate (%)=(A-B)/A×100,
where
A is the lesion diameter recorded in untreated control, and
B is the lesion diameter in the infected tomato fruit treated with the antagonists (
Sadfi et al., 2008). For each treatment, twelve tomato leaves were assayed (three leaves as one replicate),) and the experiment was repeated at least three times.
Identification of antagonistic bacteria strain EB-28
The antagonistic bacteria strain EB-28 was phenotypically characterized on the basis of morphology, physiological tests, and chemical analysis, and identified according to the methods of Bergey’s Manual of
Determinative Bacteriology (1994) and
Hacene et al. (2004), which were based on the following phenotypic features: colony and cell morphology, motility, spore production, Gram staining, pigmentation, and growth at different NaCl concentrations of 2%, 5%, 7%, and 10% (w/v). Catalase was determined by adding 10 volumes of H
2O
2 to cell culture (18 h) on solid LB medium. H
2S production was performed according to
Clarke (1953). Nitrate reduction was assayed by adding 0.2% (w/v) KNO
3 to the liquid LB medium. Voges-Proskauer tests were performed by standard procedures (
Barritt, 1936).
16S rRNA gene sequence determination was conducted by extracting chromosomal DNA from a single colony for PCR procedure according to
Xiao et al. (2000). 16S rDNA from EB-28 genomic DNA was amplified with the primers 63F 5′-CAGGCCTAACACATGCAAGTC-3′ and 1494R 5′-GGTTACCTTGTTACGACTT-3′. The PCR amplification condition was as follows: denaturation at 94°C for 5 min followed by 35 cycles at 94°C for 40 s each, at 56°C for 40 s, and at 72°C for 1.5 min, with another extension at 72°C for 10 min (
Guo et al., 2007). The PCR products were purified using a UNIQ-10 gel extraction kit (Shanghai Sangon Biological Engineering Technology & Services Co., Ltd.) and identified by horizontal electrophoresis on 1.2% agarose gel, and then sequenced with an ABI 3100-Avant Genetic Analyzer (Applied Biosystems). The DNA sequence homology searches were performed using the online BLAST search engine in GenBank (available at: http://www.ncbi.nlm.nih.gov).
Statistical analyses
The results of the experiments were analyzed with DPS (Data Processing System 3.01) software. The data presented were the mean values of the three replications. The data were subjected to variance analysis, and one-way ANOVA or t-test was used to estimate the significance of differences between mean values (P<0.05).
Results
Screening of effective antagonists in vitro
The results of the in vitro screening revealed that seven out of 238 endophytic bacterial isolates (EB-7, EB-11, EB-15, EB-19, EB-28, EB-112, and EB-122) significantly reduced the mycelial growth of B. cinerea by forming an inhibition zone (Table 1, Fig. 2(c)). Isolates EB-15, EB-28, and EB-122, which are the representative isolates from S. tuberculata, L. esculentum, and D. dasycarpus, were selected from the in vivo test.
Effective antagonist tests in vivo
Isolates EB-15, EB-28, and EB-122, which are the representative isolates from S. tuberculata, L. esculentum, and D. dasycarpus were tested on cucumber seedling cotyledons and detached tomato leaves. The most effective was the EB-28 that was isolated from the tomato stem, with a percentage of gray mold reduction of 44.8% (cucumber cotyledon) and 52.4% (tomato leaves) (Fig. 2 and Fig. 3).
Characterization and identification of EB-28
The optimum growth occurred in medium containing 2% salt (w/v; Table 2). The EB-28 isolates were Gram-positive, motile endospore-forming rods. The spores were spherical or sometimes ellipsoidal and were located at subterminal positions. The spores survived heating at 80°C for at least 10 min. Colonies of EB-28 were 1-2 mm in diameter after being cultivated for 48 h on LB medium, and circular, smooth, and cream-pigmented (Fig. 4). EB-28 was identified as the member of the genera Bacillus from its morphological, biochemical, and physiological characteristics (Table 2).
By the 16S rDNA sequence analysis, EB-28 showed 99.1% similarity to B. subtilis EF488979. Therefore, it was identified as B. subtilis (Fig. 4).
Discussion
The aim of this research was to isolate endophytic bacteria with an ability to control or at least reduce the pernicious effects of the pathogen Botrytis cinerea Pers. The in vitro pre-screening test of dual culture allowed us to select three endophytic bacterium strains. Among the three tested trains, B. subtilis EB-28 showed potential as a biocontrol agent for gray mold in vivo and may be used in biologically controlling the phytopathogenic fungus B. cinerea. Further investigation is needed to verify the effectiveness of EB-28 in field and long-term storage conditions.
It is shown previously that
Bacillus species was the predominant bacterium of all the isolated endophytic bacteria. When Moundt and
Hinckle (1976) isolated the endophytic bacteria from the surface-sterilized ovules and seeds of 27 plant species, the
Bacillus species occurred more frequently than any other species of single bacterial genus, accounting for almost one-third of all bacteria isolated. In the work of
Walker et al. (1998), 72 of the 92 isolates obtained from the spermospheres of peas and dwarf French beans were found to be
Bacillus species. Recent studies undertaken by
Sadifi et al. (2001, 2002), showed the successful biocontrol of the
Bacillus species in the inhibition of the post-harvest disease of potatoes induced by
Fusarium sambucinum under storage conditions. In our research, more than half of the isolated endophytic bacterium strains were identified as
Bacillus species according to their morphological characteristics (data not shown). This special microbial flora should be exploited more extensively in the biological control of phytopathogenic fungi.
The low rate of polymorphism in the rDNA transcription unit allowed the characterization of the rDNA of each species using only a few specimens and made this DNA useful for interspecific comparisons. In addition, the different coding regions of the rDNA repeats usually showed distinct evolution rates. As a result, this DNA could provide information about almost any systematic level (
Hillis and Dixon, 1991). The comparison of 16S rDNA sequences was one of the most powerful tools for the classification of microorganisms (
Wu et al., 2006). In our study, seven endophytic bacterium isolates from
L. esculentum Mill.,
S. tuberculatae, and
D. dasycarpus effectively inhibited the mycelial growth of
B. cinerea, and one strain encoded EB-28 significantly inhibited the lesion extension of gray mold on the cucumber seedling cotyledons and detached tomato leaves. The characterization of EB-28 was performed by conventional methods using morphology, physiological tests, and 16S rDNA sequence analysis. It was identified as
B. subtilis. Further efforts are needed to exploit
B. subtilis EB-28 and other endophytic bacterium isolates for commercial production and application under storage and greenhouse conditions.
The
Lycopersicum esculentum Mill.,
Speranskiae tuberculatae (Bge.) Baill, and
Dictamnus dasycarpus Turcz. used to isolate endophytic bacteria in the present work were selected from dozens of plant species during pre-screening experiments (unpublished data). The tomato (
L. esculentum) is an important host plant of
B. cinerea. Endophytic bacterium strains isolated from tomatoes are considered to have the potential to be used as a biocontrol agent against tomato gray mold in field and post-harvest conditions.
S. tuberculatae and
D. dasycarpus are two Chinese traditional medicinal herbs.
S. tuberculatae is useful in treating all types of acne when used in conjunction with
Herba Menthae,
Radix Gentiana, and other herbs of the same family (
Li et al., 2000).
Dictamnus (
Dictamnus dasycarpus; baixianpi) is one of the most commonly used Chinese herb for the treatment of eczema (
Du et al., 2005).
S. tuberculatae and
D. dasycarpus have been found to have antifungal activity against some plant pathogens, such as
Rhizoctonia solani,
Alternaria longipes, and
Curvulavia lunata (
Du et al., 2005;
Huang et al., 2007) and are regarded as potential biocontrol agents to plant diseases. In this study, four endophytic bacterium isolates from
S. tuberculatae and two isolates from
D. dasycarpus showed significant antagonistic effects against
B. cinerea in vitro, while the inhibitive effects of these two plants against
B. cinerea were still unclear. An interesting question is whether combined biological control agents made up of endophytic bacteria and plant extracts would have a stronger biocontrol effect against tomato gray mold or other plant diseases.
Higher Education Press and Springer-Verlag Berlin Heidelberg
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