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Screening the Antibacterial Activities of Streptomyces Extracts against Phytopathogens Xanthomonas oryzae pathovar oryzae, Xanthomonas campestris pathovar vesicatoria, and Pectobacterium carotovorum pathovar carotovorum

Xanthomonas oryzae pv. oryzae (Xoo), X. campestris pv. vesicatoria (Xcv), and Pectobacterium carotovorum pv. carotovorum (Pcc) are the causative agents of bacterial blight in rice, bacterial spot in pepper, and bacterial soft rot in carrot and cabbage, respectively. To isolate novel microbial extracts with antimicrobial activities against these bacteria, approximately 5,300 different Streptomyces extracts were prepared and tested. Microbial cultures from various Streptomyces strains isolated from the Jeju Island, Baekam, Mankyoung river, Jiri mountain etc. in Korea were extracted into three different factions -secreted hydrophobic, secreted hydrophilic, and mycelia- using ethyl acetate, water, and methanol. Initially, 34, 29, and 10 extracts were selected as having antibacterial activities against Xoo, Xcv, and Pcc, respectively. Extracts 1169G4, 1172E9, and 1172E10 had the highest growth inhibition activities against both Xoo and Xcv, and extracts 1151H7 and 1152H7 showed the highest growth inhibition activities against Pcc.

Rice, pepper, and cabbage are the first, second, and third most cultivated crops, respectively, in Korea. Xanthomonas oryzae pv. oryzae (Xoo) and X. campestris pv. vesicatoria (Xcv) are the causal agents of bacterial blight in rice and bacterial spot in pepper, respectively (Yang et al., 2005;Adhikari et al., 2010). Pectobacterium carotovorum pv. carotovorum (Pcc) causes soft rot in a wide range of plants, including cabbage (Mole et al., 2010). Because these bacteria cause severe crop damage and reduce yield, prevention and treatment of these diseases have been the focus of intense research in both the academy and industry (Khush, 1997). In 2005, the whole genomes of Xoo and Xcv were sequenced (Frank Thieme, 2005;Lee et al., 2005). The emergence of resistance against Pcc was reported in 2007 (T. Luzzatto, 2007).
Chemical pesticides are routinely used to control plant pathogens in agricultural fields. However, recently, indiscriminate usage of chemical pesticides has raised many concerns, including environmental toxicity (Vivek K. Bajpai, 2010) and the possibility of generating antibiotic-resistant microbes. Therefore, biopesticides made from secondary metabolites from microbes or plants are being preferred over chemical pesticides.
The main objective of this study was to isolate novel antimicrobial Streptomyces extracts against the phytopathogens mentioned. Streptomyces species are ubiquitous in the soil, and play an important ecological role in the turnover of organic material. Furthermore, the diverse secondary metabolites produced by Streptomyces species are good sources of novel bioactive compounds. To date, more than half of the world's antibiotics (over 50 different antibiotics) have been isolated from Streptomyces. In this study, we prepared and screened 5,328 Streptomyces extracts, and tested their antimicrobial activities against Xoo, Xcv, and Pcc.

Materials and Methods
Strains and Culture Conditions. X. oryzae pv. oryzae KACC10331 (Xoo), X. campestris pv. vesicatoria KACC11127 (Xcv) and P. carotovorum pv. carotovorum KACC10227 (Pcc) were provided by the Korea Agriculture Culture Collection (KACC). Xoo, Xcv, and Pcc were grown in YPD (1.0% yeast extract, 2.0% peptone, and 2.0% dextrose) rich media at 28 o C. Streptomyces strains, collected from various regions of the Republic of Korea, including Jeju island, Baekam, Mankyoung river, Jiri mountain etc. (S-Table 1), were maintained at the Extract Collection of Useful Microorganisms library at the Myoungji University, and grown at 28 o C on GSS medium (1.0% soluble starch, 2.0% glucose, 0.1% beef extract, 0.4% yeast extract, 0.2% NaCl, 0.025% K 2 HPO 4 , and 0.2% CaCO 3 at pH 7.2), Bennett's medium (1.0% glucose, 0.1% yeast extract, 0.2% bacto-peptone, and 0.1% beef extract), or DYC medium (2.5% dextrin, 1.2% dry yeast, 2.0% corn starch liqueur, 0.1% NaBr, and 0.1% CoCl 2 at pH 7.0). Preparation of the Streptomyces extracts. For preparation of Streptomyces extracts library, one gram of soil were suspended in 10 milliliter of 0.85% NaCl solution and serially diluted. The diluted soil suspension were spread on Humic acid-vitamin (HV) agar medium (0.1% humic acid, 0.05% Na 2 HPO 4 , 0.071% KCl, 0.005% MgSO 4 ·7H 2 O, 0.001% FeSO 4 ·7H 2 O, 0.002% CaCO 3 , 0.2% B-Vitamin solution, 1.8% agar at pH 7.2) and incubated at 28 o C for 14 days. The colony with representative actinomycetes were selected and identified by 16S rDNA sequence. Then Streptomyces strains were cultured in three different media: GSS medium for nutrient-rich condition, Bennett's medium for moderatenutrition condition, and DYC medium for nutrient-poor condition. Three extract fractions were prepared from each Streptomyces culture broth. 30 mL of the culture broth were centrifuged at 5,000×g for 10 min. Mycelia were collected as a pellet, and extracted with 3 mL of methanol. This fraction was called "mycelia isolated from supernatant (MIS)". After pelleting the mycelia, the supernatant was further extracted with 30 mL of ethyl acetate and dried. The resulting dried extract was dissolved in 3 mL of methanol. This was called the "secreted hydrophobic (SHB) fraction". The remaining ethyl acetate-insoluble fraction was called the "secreted hydrophilic (SHL) fraction". Thirty microliter of each fraction was distributed into 96-well plate and dried completely before further use. The dry weights were measured to enable accurate calculation of GI50 (dose required to cause 50% growth inhibition). Naming the Streptomyces extracts. All extracts were represented as "0000X0." The four digits, preceding the alphabet "X," refer to the plate number, which is named according to the region from where the sample was collected. The alphabet X refers the row in which the sample was present (numbered from top to bottom) and the last digit represents the column (numbered from left to right). For instance, sample 1172H2 is located in the second column from the left and at the bottom row of the 96-well plate numbered 1172. In addition, the last digit of the extract's name represents the extract-type: 2, 5, and 8 represent SHL; 3, 6, and 9 represent MIS; and 4, 7, and 10 represent SHB. Antimicrobial activity test. The antimicrobial activities of the extracts were estimated by the growth inhibition test using a single dose in two steps. In the first round of screening, all the extracts were stored in columns 2-10 of the 96-well plate. Columns 1, 11, and 12 contained the control (medium only), bacterial culture, and To measure the antibacterial activity of each extract, we calculated the growth inhibition rate (GIR) at 20 h for Xoo and Xcv and at 12 h for Pcc using the following formula:

GIR=(1[CB]/[AB])×100
where A was the A 600 value of the bacterial culture with 6% DMSO; B =0.2, which was the A 600 value of the bacterial culture when the extract was added; and C was the A 600 value of the bacterial culture after treatment with the extract. For the second growth inhibition screen, the extracts selected from the first assay were tested three times using the same procedure. The amount of growth inhibition induced by each extract was measured at five different dosages (1, 10 −1 , 10 −2 , 10 -3 , and 10 −4 dilutions) and compared with the controls. From this, the GI50 was calculated. All the tests were carried out in triplicate.

Results
Preparation of extracts from Streptomyces. Streptomyces strains produce diverse physiological secondary metabolites depending on the culture conditions. To generate various secondary metabolites from each Streptomyces strain, three culture media with different nutritional levels were used to grow the Streptomyces cells. In addition, three extract fractions were prepared for each Streptomyces culture. The MIS was separated from the supernatant; SHBs were extracted from the supernatant fraction using ethyl acetate; and SHLs were obtained from the residual water layer. In total, 5,328 extracts were prepared and screened for their antimicrobial activities against the three plant pathogens, Xoo, Xcv, and Pcc. First round of screening for antimicrobial activity. Extracts showing more than 50% cell growth inhibition activity were considered antimicrobial extracts; extracts 34, 29, and 10 were selected because they exhibited antimicrobial activity against Xoo, Xcv, and Pcc, respectively. These three extracts constitute 0.64, 0.53, and 0.19% of all the extracts tested in the study ( Fig. 1 and Table 1).
The GI50s of the five antimicrobial extracts were measured by serial dilution of the extracts (Table 2). 1172E9 (MIS) and 1172E10 (SHB) showed the highest antibacterial activities against Xoo and Xcv ( Fig. 4 and 5). In particular, 1172E10 (SHB) showed a GI50 value of less than µg·mL -1 . 1152H7 (SHB) had a slightly Fig. 3 The growth curve of (A) Xoo, (B) Xcv, and (C) Pcc upon treatment with the screened antibacterial extracts in the second round of growthinhibition screening. The extracts were added at the early exponential growth stage (absorbance at 600 nm [A 600 ]=0.2, time=0) of each bacterial culture, and bacterial growth was monitored for an additional 12-15 h by measuring A 600 . Untreated bacterial culture was used as the control sample.

Discussion
Antibiotics are secondary metabolites that inhibit the growth of other microbial organisms. Growing incidences antibiotic-resistance among deadly pathogens has increased the need to discover novel antibiotics. Over the last few decades, new technologies for drug discovery have been developed, such as high throughput screening  and combinatorial chemistry. Moreover, sequencing of pathogen genomes has led to the identification of new drug targets, and many chemical compounds have been synthesized by combinatorial chemistry and screened using cell-free assay systems and highthroughput screening systems. However, most screened compounds do not penetrate the pathogen to reach the target, or are easily degraded in vivo. Despite these technological advances, only five new classes of antibiotics have been introduced for human use (Talbot et al., 2006). Therefore, many studies are now focusing on screening secondary metabolites from microbes, especially Streptomyces, to identify novel antimicrobials. Streptomyces bacteria produce numerous secondary metabolites, and approximately 55% of currently used antibiotics are produced by the genus Streptomyces.
Five Streptomyces extracts showing high antimicrobial activity against Xoo, Xcv, and Pcc were screened. Of the five extracts, four (1152H7, 1151H7, 1169G4, and 1172E10) were secreted and hydrophobic, and only one extract (1172E9) was non-secreted. The secretory and hydrophobic properties of the extracts aid in the penetration of bacterial cell walls (Nikaido H, 1976;Savage et al., 2002). In particular, 1169G4, 1172E9, and 1172E10 were effective against both Xcv and Xoo. The genus Xanthomonas belongs to Proteobacteria, and it causes a variety of diseases in numerous crops. The extracts identified in this study may have applications as a novel antimicrobial drug against Xanthomonas, including Xoo and Xcv.