Streptomyces nobilis C51 Suppresses Gray Mold Caused by Botrytis cinerea in Tomato

This study sought to develop a biological control agent against gray mold disease caused by Botrytis cinerea in tomato using a strain of Streptomyces, which showed significant antagonistic activity against B. cinerea. Such strain (C51) was screened by dual culture method from a total of 78 actinomycetes and was identified as Streptomyces nobilis by morphological, biochemical and molecular analysis. The influence of different environmental conditions, such as temperature degree, pH range and medium type on the expression of the antifungal activity was thoroughly examined. The metabolites involved in the antagonistic action of C51 strain showed to be produced independently by the presence of B. cinerea. We also found that the culture filtrate of C51 potently inhibited the mycelial growth of B. cinerea, and morphological changes such as hyphal swelling and Original Research Article Jiang et al.; BMRJ, 16(6): 1-13, 2016; Article no.BMRJ.27284 2 abnormal shapes were observed using culture filtrates. The antifungal activity of the culture broth was stable across a broad range of temperatures (28-121°C) and pH (pH 2-12), suggesting a wide range of applications of Streptomyces nobilis C51. The results of greenhouse test showed that Streptomyces nobilis C51 had the same disease inhibition effect as well as azoxystrobin against B. cinerea. The current observations could support the application of Streptomyces nobilis C51 as a promise biocontrol agent.


INTRODUCTION
Since Micheli erected the genus Botrytis in 1729, it has become a widely known group of fungi causing diseases on economically important plants [1][2][3][4]. B. cinerea is one of the most destructive plant pathogens in this genus which attacks a wide variety of plants (over 200 plant species) and causes severe diseases worldwide. B. cinerea also spoils agricultural products after harvest since it can be active at very low temperatures [5][6]. This fungus infects flowers, fruits, and vegetative tissues in tomato and leads to severe loss in yield and quality.
Conventionally, B. cinerea are controlled by chemical fungicides such as benzimidazole and dicarboximide. However, this is becoming less acceptable because it conflicts with public concern for fungicide residues and also increases the potential for the build-up of resistance in B. cinerea to fungicides [7]. Consequently, development of high-efficient and environment friendly methods for preventing gray mold in tomato is an increasing demand from consumers and officials [8]. In this context, biological control through the use of antagonistic microorganisms has emerged [9].
Several antagonistic microbes including the genera Bacillus subtilis [10][11], non-pathogenic Fusarium [12], Trichoderma [13], Pseudomonas [14][15] and Streptomyces [16] are used as biocontrol agents. However, each antagonistic microbe possesses undesirable features for its use as biocontrol agent. Pseudomonads is unable to produce the resting spores which hinders their commercial use [17]. Bacillus strain has rich genes to produce a variety of antibiotics, but only a small part of genes can synthesize the antibiotics that can be secreted under natural conditions [18]. The non-pathogenic Fusarium strains suppress the pathogens depending on its competition of nutrients with pathogens so that a large amount of Fusarium need to be applied in practice.
Among various fungal and bacterial biocontrol agents, soil bacteria from genus Streptomyces are proved to be the most frequently used against various plant diseases. The antagonistic activity of Streptomyces is usually related to the production of antibiotics, secretion of extracellular hydrolytic enzymes, competition and hyperparasitism [19]. El-Abyad et al. [20] reported that the culture filtrate of either S. pulcher or S. canescens significantly inhibited spore germination, mycelial growth and sporulation of Fusarium oxysporum f. sp. lycopersici (the cause of Fusarium wilt), Verticillium albo-atrum (the cause of Verticillium wilt) and Alternaria solani (the cause of early blight) in tomato respectively. The in vivo studies demonstrated that coating tomato seeds with spores of the antagonist was the most effective in controlling the tomato diseases concerned [20]. Chemical fungicides have often been replaced with Streptomyces sp. to control pathogens [21]. These data indicated that Streptomyces species could be used as biological control agents effectively. However, the data related to the antagonistic ability of the Streptomyces metabolites against B. cinerea are limited.
In this study, a total of 78 actinomyces were screened using selective media from agricultural soils of Cangzhou, China. A S. nobilis strain named as C51 exhibiting the highest antagonistic activity against B. cinerea were isolated. The culture conditions for this strain were optimized and stability of the bioactive compounds at different temperature and pH was determined. Moreover, the antifungal activity of medium extracts from solid and broth culture of S. nobilis C51 were investigated. The antagonistic mechanism of the strain was discussed. In vivo experiments revealed that culture broth of S. nobilis C51 was capable to control gray mold disease in tomato effectively. This information will be useful to design strategies to enhance crop protection against Botrytis diseases.

Fungal Strains and Culture Conditions
B. cinerea was used as the tested fungal pathogen which was cultured on Potato Dextrose Agar (PDA) at 22°C for 10 days. The spores were harvested by flooding the surface of 10-day-old cultures with distilled water and the inoculum concentration used was 1×10 6 spores /mL.

Soil Sampling and Isolation of Actinomycetes
Samples of soil were collected from orchards and agricultural fields in different parts of Cangzhou, China. 5 g soil sample was mixed with 45 mL of sterile water and shaked vigorously for 10 min. The soil mixture was serially diluted in sterile distilled water. 100 µL of 10 4 dilution was spread onto Gause's Synthetic Agar Medium (soluble starch 20.0 g/L; KNO 3 1.0 g/L; NaCl 0.5 g/L; MgSO 4 ·7H 2 O 0.5 g/L; FeSO 4 ·7H 2 O 0.01 g/L; KH 2 PO 4 0.5 g/L; agar 18.0 g/L), which was supplemented with 50 µg/mL of filter-sterilized K 2 Cr 2 O 4 to inhibit bacteria and fungus growth. Plates were incubated at 28°C for 10~14 days. Subsequently, colonies with different morphological appearances were selected from the countable plates and re-streaked onto a new plate of the same media to obtain pure colonies. For long term storage, the spores' suspensions of actinomycetes were maintained in 30% (w/v) glycerol at -20°C.

Dual Culture Bioassay
The antagonistic activity of the actinomycetes strains were determined using agar plates [17]. Nine centimetres Petri plates containing potato dextrose agar (PDA) were inoculated with 6 mm mycelial disks of either B. cinerea (4-day-old culture) and actinomycetes strain (7-day-old culture) 3.0 cm apart from each other. Plates inoculated with B. cinerea alone served as control. The plates were incubated at 22°C for 4 days, then diameter of inhibition zones was measured. The antifungal activity was calculated according to Kanini et al. [22]. The experiment was performed three times with five replicate PDA plates.

Phenotypic Characteristics of C51 Strain
To determine cultural characteristics of C51 strain, we have grown it on various ISP medium [23] at 28°C for 14 days. The color of aerial and substrate mycelia including soluble pigment were determined. The growth situation at different medium were determined by the following standard: Relative value of growth (R) = ‫=ݖܴ(‬ radius of streptomycete colony on various medium and ܴ str = radius of streptomycete colony on Gause's Synthetic Agar Medium). R = ~0.5 Poor; R = 0.5~0.8 Moderate; R = 0.8~1.2 Good; R = 1.2~ Excellent.
Biochemical tests including starch and gelatin hydrolysis, voges-proskauer, citrate utilization, indole, methyl red, gelatin and nitrate reduction were determined by standard methods [24]. Utilization of different carbon sources such as glucose, starch, mannitol, fructose, soluble corn starch and maltose, and nitrogen sources namely arginine, tyrosine, glutamic acid, aspartic acid, NaNO 3 and (NH 4 ) 2 SO 4 were also tested.

Taxonomy of C51 Strain
For molecular identification, the 16S rDNA gene of C51 strain was amplified using the primers 27F and 1492R [25]. PCR amplifications were performed as following: initial denaturation at 94°C for 2 min, 35 cycles of 94°C (60 sec), 50°C (60 sec), and 72°C (2 min) followed by final extension at 72°C for 10 min. Amplified PCR products were separated by gel electrophoresis on 1% (w/v) agarose gel and then purified using Extract PCR kit (TaKaRa Biotechnology Co., Dalian, China). Purified PCR product of approximately 1,400 bp was sequenced at Life Technologies Biotechnology Company, Shanghai, China. For the detection of closest relatives, the 16S rDNA gene sequences of C51 strain of were identified using the BLAST program (http://www.ncbi.nlm.nih.gov/Blast/). Sequence data was compiled using the MEGA 6.05 software and aligned with sequences obtained from the GenBank (http://www.ncbi.nlm.nih.gov/) databases using the DNAMAN aligning utility. Phylogenetic analysis was performed using the neighbour joining method implemented in MEGA 6.05.

Effects of Culture Conditions on Antagonistic Activity of S. nobilis C51
The agar plate antagonism bioassay of C51 strain was performed on five different solid media including PDA, Arginine Glycerol Salts (AGS), Nutrient Agar (NA), Czapek Agar (CzA) and Yeast extract-malt extract medium (YEME). The antagonistic activity of C51 at different temperature degrees (22, 24, 26, 28 and 30℃) and different pH (5, 6, 7, 8, 9 and 10) was tested using YEME medium. The C51 strain was inoculated and incubated at different culture conditions for 4 days. The antifungal activity following exposure to various temperatures and pH conditions was measured as above.

Extraction of S. nobilis C51 Metabolites from Solid Cultures
S. nobilis C51 was grown on YEME with pH 7 and incubated at 28°C since these were selected as the best conditions for high antifungal activity expression. After 7 days, the medium around S. nobilis colony in a radius of 2 cm was cut off and crushed into slurry. The slurry was centrifuged at 6,000 g for 30 min and the supernatant was collected by tips. To determine the antifungal activity, 50 µL of the extracted material from medium were placed into wells on PDA plates, formed using a cork borer 6×5 mm (diameter×depth) that were inoculated with the fungus.
In addition, the inhibition zones of co-cultures of the selected S. nobilis and B. cinerea on YEME plates were removed and mixed. The supernatant was obtained from the slurry by centrifugation as described above. After filtration, 50 µL of the supernatant was placed into wells on PDA agar plates which were inoculated with the fungus.

Fermentation of S. nobilis C51
S. nobilis C51 was cultured in a 250 mL Erlenmeyer flask containing 50 mL of YEME at 28°C with 180 rpm for 10 days. The culture broth of S. nobilis C51 was centrifuged at 6,000 g at 4°C for 20 min and the supernatant were sterilized by filtration using the membrane of 0.22 µm in pore size. The resulted crude extracellular metabolites were stored at 4°C.

Antifungal Activity of the Culture
Broth of S. nobilis C51 6 mm mycelial disks was taken from a freshly growing colony of B. cinerea and placed on PDA plates (9 cm) which contained the indicated dilution of the cell-free suspension of S. nobilis C51. They were kept in incubator at 22°C for 3-4 days. Diameters of the fungal colonies were measured and recorded. Inhibition rate was calculated by formula described as Paul [26].
To further evaluate the antifungal effect of the extracellular metabolites, fungal hyphal morphology which incubated on the PDA plate with and without cell-free culture broth of S. nobilis C51 was observed under a light microscope (Leica, Germany). Abnormalities in the morphologies of hyphae was observed and photographed with a digital camera attached to the photoport of the microscope.

Assay for the Temperature and pH Stabilities of Antifungal Activity of S. nobilis C51 Culture Broth
For the thermal stability test, the culture broth was exposed to 28, 40, 50, 60, 70, 80, 90, 100 and 121°C for 20 min and cooled down on ice.
For the pH stability test, the culture broth was adjusted to pH 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12 for 24 hours and adjusted to the original pH. The treated culture filtrates (1 mL) were mixed with 19 mL warm molten PDA and their antifungal activities against B. cinerea was assessed by radial growth inhibition assay. Fungal growth was measured after 4 days of incubation and the percentage of growth inhibition was calculated.
Residual activities against B. cinerea were measured as relative to the activity of 28°C.

Greenhouse Studies
To prove the inhibitory effects of S. nobilis C51 under greenhouse conditions (photo period of 12 h of light,12 h of darkness, light intensity of 100 µmol·m 2 ·s −1 and temperature of 22 ± 2°C), a greenhouse experiment was undertaken. The suspension of S. nobilis C51 was diluted 0, 5, 10 and 20 time(s) with sterilized water and sprayed onto whole tomato plants (> 15 cm in height). The chemical fungicides azoxystrobin and water were used as positive and negative controls respectively. The solution of B. cinerea spores (1 × 10 6 spores/mL) were sprayed onto whole plants 1 day before treatments with 0, 5, 10 and 20 time(s) diluted suspension of S. nobilis C51. Each agent was tested in triplicate. All treated plants were covered with transparent plastic bags to prevent accidental dryness. 7 days after inoculation, the disease severity of gray mold was recorded. The disease incidence of the gray mold infected plants was divided into 5 scales. The gray mold development on each plant was recorded using 1-5 scale developed, where 0 = no visible gray mold development, 1 = 1-5%, 3 = 6-15%, 5 = 16-25%, 7 = 26-50%, and 9 =≥51% of the leaf surface covered with gray mold. A, B, C, D and E are the number of leaves corresponding to the scale 9, 7, 5, 3, and 1, respectively, and F is the total number of assessed leaves. Disease severity was calculated using the following formula [27].

Statistical Analysis
Antagonistic effects of S. nobilis C51 against B. cinerea were analyzed based on completely randomized design (CRD) with three replications. Statistical analysis was performed with SPSS version 17.0 software. The various data sets were analyzed by one-way analysis of variance (ANOVA).

Actinomycete Isolation, Identification and Antifungal Bioassay
A total of 78 actinomycetes were isolated using selective media. Judged by the radius of inhibition, 18 isolates with antifungal activity against B. cinerea were screened ( Table 1). The radius of inhibition of the 18 isolates were from 1.57 ± 0.26 mm to 11.07 ± 0.25 mm. The total percentage of antagonistic isolates was 23%. Among 18 isolates the strain C51 exhibited the highest antifungal activity with 11.07 ± 0.25 mm radius of inhibition (Fig. 1).  It was observed that the upper colonial appearance of C51 strain on ISP 2 medium was purplish white without soluble pigment production ( Table 2). The biochemical identification revealed that the C51 strain was positive for citrate, H 2 S, and starch hydrolysis but negative for Vogesproskauer, indole and nitrate reduction, and utilized glucose and mannitol as carbon sources (Table 3, Fig. 2). The data obtained from morphological and biochemical analysis demonstrated that C51 strain might be Streptomyces species. To further identify this strain, we performed 16S rRNA gene sequence analysis (GenBank accession Number KM025197; Fig. 3). The results grouped the C51 strain to the species of S. nobilis (99% identity with the 16S rRNA sequence of the closest phylogenetic relative) (Fig. 4).

The Effect of Different Artificial Condition on Antagonistic Activity of S. nobilis C51
Giving that the culture medium plays a crucial role in regulating the antagonistic feature of microbes, we used five kinds of medium to cultivate C51 strain and evaluated the effects of medium type on antagonistic activity of S. nobilis C51. The highest antagonistic activity was obtained when S. nobilis C51 was grown on 9 7 5 3 100 9 A B C D E F + + + + × Orange-red Dark red Good YEME medium. The antagonistic activity of the strain grown on NA medium was slightly less than that grown on YEME medium (Fig. 5a). Notably, no antagonistic activity was detected when cells were grown on CzA medium (data not shown).
Subsequently, the optimal temperature and pH were determined. The results showed that the maximal antagonistic activity was obtained when C51 strain was cultivated in YEME medium at 28℃ with pH 7.0 ( Figs. 5b and 5c).

The Antagonistic Activity of Extract of S. nobilis C51 Metabolites from Solid Cultures
To investigate whether the antagonistic activity of S. nobilis C51 is induced by the presence of pathogen, we extracted the metabolites from solid medium of C51 strain in presence or absence of B. cinerea and compared the antifungal activity of the extracts. The results revealed that the extract from the agar inhibition zones of S. nobilis C51 with the presence of B. cinerea could strongly suppress the growth of B. cinerea (Fig. 6). Likewise the same antifungal activity was observed from the extract of the solid culture of C51 isolate without the presence of B. cinerea. These data implicated that the presence of target fungus might not be the requirement for the induction of antifungal activity of S. nobilis C51.

Antifungal Activity of the Culture Broth of S. nobilis C51
Based on the results described above, it has been known that S. nobilis C51 could secrete the bioactive component extracellularly. Thus, we further assess the effects of cell-free culture broth of C51 strain on the growth of B. cinerea. Plant pathogenic fungi was inoculated on the plate containing culture broth of S. nobilis C51 with different dilutions. B. cinerea was inhibited on the PDA with 5-fold and 10-fold dilutions of culture broth of S. nobilis C51 completely (Fig. 7). The inhibition rates with 20-fold and 80-fold dilutions were 70.8 ± 0.41% and 14.5 ± 0.95%, respectively. The swollen balloon-like structure was found when B. cinerea was cultivated for 3 days on PDA plate with 20-fold dilutions cell-free culture filtrate (Fig. 8b), while the hypha of B. cinerea exhibited a normal morphology on PDA plate (Fig. 8a).

Thermal and pH Stability of S. nobilis C51 Culture Broth
To explore the environmental adaptation of the bioactive components, the thermal and pH stability of culture broth of strain C51 was investigated. The culture broth of S. nobilis C51 was exposed to various temperatures for 20 min, and the remaining activity was measured. The antifungal activity of heat-treated culture broth against B. cinerea was highest at 28°C (100%), and more than 80% of the activity was maintained at 40, 50, 60, and 70°C. Notably, after treatment with 121 o C for 20 min, 76.4 ± 0.4% of activity remained ( Table 4).
The antifungal activity of pH-adjusted culture broth against B. cinerea was highest at pH 6 (100%), and more than 85% of the activity was maintained at pH 2, 3, 4, 5, 7 and 8. At pH 12, 48.6±1.43% of activity still remained. These results demonstrated that the antifungal activity of the culture broth of strain C51 was stable at a wide range of temperature and pH (Table 5).

Greenhouse Experiment
To determine if this inhibitory efficacy of S. nobilis C51 works well under greenhouse conditions, a greenhouse experiment was done. When 0, 5, 10 and 20 time(s) diluted culture broth were used, the disease severity were 27.9 ± 1.07%, 40.49 ± 1.83%, 43.09 ± 2.78% and 49.51 ± 5.27% respectively. All treatments could control the disease efficiently and there were less lesion on the leaves of treatment than that of water control. The percentage of disease inhibition rate for the stock solution of S. nobilis C51 was 72.63±0.33%, and azoxystrobin was 72.51±0.80% (Table 6). The results suggested that S. nobilis C51 can be considered as an efficient agent as semilar as chemical fungicide for B. cinerea treatment.

DISCUSSION
In this study, an antagonistic strain against B. cinerea was isolated from soil and was named as S. nobilis C51. In vitro and greenhouse experiments indicated that S. nobilis C51 could be used as a good candidate for the biocontrol of gray mold in tomato caused by B. cinerea. In nature, plant coexist and interact with naturally colonized microflora including plant pathogens and beneficial microbes, which make the natural environment become the best source to screen antagonistic microorganisms. In this work, we collected soil samples from the farmland where the soil-borne and air-borne disease in crops occurred seriously and screened the microbial strains which have the ability to control B. cinerea. We focused on screening the Streptomyces genus by using the selective medium since several strains of this genus have been used as biocontrol agents for a wide variety of fungal pathogens [28][29]. We obtained a strain encoded C51 showing the highest antagonistic activity against B. cinerea in the dual culture experiment. The morphological, physiological and molecular analysis assigned this isolate to be S. nobilis. Our results suggested a role of sampling area for screening the targeted antagonistic microorganism.

Recently, Kanini et al. screened the indigenous
Streptomyces with antifungal activity with high percentages from the area which is characterized by its high temperature and low humidity [22,30]. This environment may exert the stress on the indigenous populations of Streptomyces and possibly leads to the prevalence of microorganisms with antagonistic properties. As like, the situations of frequently occurrence of soil-and air-borne disease impose the selective pressure on the local population of Streptomyces and might confer them the ability to suppress the growth of the fungal pathogens.   Recently, Kanini et al. [30] found that the extracts from the agar inhibition zone of the isolated S. rochei ACTA1551 could strongly suppress the growth of pathogen Fusarium oxysporum, meanwhile the same antifungal activity was observed from the extracts of the solid agar around the colony of S. rochei ACTA1551 without the presence of F. oxysporum. They, therefore, speculate that the antifungal activity of Streptomyces is fungus independent. Our results showed that the production of antifungal compounds by S. nobilis C51 does not require the target fungus and the expression of antifungal activity of S. nobilis C51 might be constitutive. The cell-free culture broth of S. nobilis C51 has strong ability to suppress the growth of B. cinerea further supports this conclusion. However, the induction pattern of antifungal activity for the Streptomyces might be fungal pathogen specific. For instance, Kanini et al. observed the antifungal activity only in the solid culture extracts but not in the liquid culture supernatant of S. rochei ACTA1551 when another soil-borne fungal plant pathogen Rhizoctonia solani was used as tested pathogenic fungus [30]. In addition, the intrinsic difference between solid and liquid culture including oxygen availability, the nutrient limitation and metabolitic intense may contribute to biosynthesis of antifungal substance in Streptomyces [31][32].
The antifungal activity of Streptomyces is usually related to the production of antifungal compounds and the extracellular hydrolytic enzyme such as chitinases, β 1-3 glucanases [33][34]. S. violaceusniger YCDE9 was reported not only to produce the extracellular hydrolytic enzymes but also to excrete three antifungal compounds [35]. Prapagdee et al. found that the extracellular hydrolytic enzymes in the exponential culture filtrate and secondary thermostable compounds in the stationary culture filtrate of S. hygroscopicus SRA14 play an important role in the inhibition of the growth of Colletotriculum gloeosporioides and Sclerotium rolfsii [36]. In the present study, the cell-free culture broth of S. nobilis C51 still retained 76.4% of antifungal activity even after treatment with autoclave at 121°C for 20 min. We speculated that the antifungal substance produced by S. nobilis C51 is probably not a protein or an extracellular enzyme, but rather a secondary metabolite. Thus, the further separation and identification of the bioactive substance from S. nobilis C51 are required and under investigation in our laboratory. Moreover, we found that the antifungal activity in the culture broth of S. nobilis C51 had broad thermal and pH stability. These features could make it possible for the bioactive components of this strain to be used as biocontrol agent under the diverse environmental conditions. Previous study by light microscopic analysis revealed that the culture filtrates of S. hygroscopicus SRA14 caused the cellular changes in hyphal morphology including hyphal swelling, distortion and cytoplasm aggregation of fungal pathogens [36]. Joo also reported that the extracellular chitinase produced by S. halstedii AJ-7 is responsible for the abnormal hyphal morphology [37]. On the other hand, abnormal hyphal structure such as thickness and bulbous roundness of the inhibited fungal hyphae resulting from diffusible secondary compounds including antibiotics and metabolites has been previously reported [38]. In accordance with these observations, we monitored the abnormal changes in hyphal structure of B. cinerea after treatment with the culture broth of S. nobilis C51. Taken the nature of the thermal and pH stability of antifungal activity into account, the pathogenic fungal growth inhibition by the strain C51 is likely to be due to presence of extracellular metabolites which cause growth aberration, hyphal swelling and suppress the fungal growth.

CONCLUSION
In this investigation, B. cinerea has been shown to be inhibited by diluted C51 culture broth. The disease severity was lower when higher concentrations of C51 culture suspension were used to control gray mold in tomato. It means that the application of S. nobilis C51 may offer farmers an opportunity to limit the use of chemical fungicides and permit biological measures for the control of gray mold. Future research will be aimed at developing technology for the application of biocontrol agents in largescale and investigating the mechanisms of strain C51 for the control of gray mold in plants.