Isolation and identification of Serratia marcescens Ha1 and herbicidal activity of Ha1 ‘pesta’ granular formulation

Journal of Integrative Agriculture 2015, 14(7): 1348–1355 Available online at www.sciencedirect.com

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RESEARCH ARTICLE

Isolation and identification of Serratia marcescens Ha1 and herbicidal activity of Ha1 ‘pesta’ granular formulation YANG Juan*, WANG Wei*, YANG Peng, TAO Bu, YANG Zheng, ZHANG Li-hui, DONG Jin-gao Mycotoxin and Molecular Plant Pathology Laboratory, Agricultural University of Hebei, Baoding 071001, P.R.China

Abstract A total of 479 bacterial strains were isolated from brine (Bohai, Qinhuangdao City, Hebei Province, China). Bioassay results indicated that 4 strains named Ha1, Ha17, Ha38, and Ha384 had herbicidal activity. And strain Ha1 had the highest effective herbicidal activity. As a result, this study aims to identify strain Ha1, characterize its physiological and biological activities, evaluate the herbicidal activity of its metabolites, and develop a ‘pesta’ formulation and assess its effectiveness on Digitaria sanguinalis. Ha1 was identified as Serratia marcescens based on 16S rDNA sequencing. This strain has a flagellum, a diameter of 0.5 to 0.8 μm, and a length of 0.9 to 2.0 μm. The indole test shows positive results, and the catalase enzyme exhibits strong positive reactions. Results further showed that the inhibitory concentration (IC50) of the crude extracts to D. sanguinalis radicula and coleoptile were 3.332 and 2.828 mg mL–1, respectively. Both the suppression of D. sanguinalis and the cell viability of the Ha1 formulation in ‘pesta’ were higher when stored at 4°C than at (25±2)°C. These results indicated that S. marcescens Ha1 can potentially be used as a biocontrol agent against D. sanguinalis. Keywords: Serratia marcescens, bioherbicide, biocontrol, pesta

1. Introduction Zea mays is one of the most important cereal crops in China. Despite its agronomic importance, however, its yields are limited by a large number of abiotic and biotic factors, including the interference of annual weeds such as Digitaria

Received 30 September, 2014 Accepted 6 March, 2015 YANG Juan, E-mail: [email protected]; WANG Wei, E-mail: [email protected]; Correspondence ZHANG Li-hui, Tel: +86-312-7520235, E-mail: [email protected]; DONG Jin-gao, Tel: +86-312-7528266, E-mail: [email protected] * These authors contributed equally to this study. © 2015, CAAS. All rights reserved. Published by Elsevier Ltd. doi: 10.1016/S2095-3119(14)60967-9

sanguinalis, and the classical methods to suppress D. sanguinalis are essentially chemical and cultural (Qiang 2001). But unfortunately, they were expensive and had caused many long-term problems (Li 2004), such as water and soil pollution, pesticide residue deposition, and emergence of herbicide resistance in many weed biotypes (Fischer et al. 2000; Vasilakoglou et al. 2000; Su 2001; Ma 2002; Yasuor et al. 2008; Mejri et al. 2013). The development of alternative pollution-free and environmentally friendly herbicides has become imperative. Biological herbicides that can control weeds are a promising and fascinating alternative to chemical herbicides. In recent years, marine microorganisms have provided a new direction for novel drug research, both domestic and overseas. Purine, tyrosine and tryptophan containing indole alkaloids were found biological activity, including anti-angiogenesis, cytotoxicity and anticancer activity (Tohme et al. 2011; Bharate et al. 2012). The marine environment is oligotrophic

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and characterized by high pressure, high salt, hypoxia, less light, and low temperature. As such, this environment provides microbial resources with metabolic pathways which significantly differ from those of terrestrial microbes (Isnansetyo and Kamei 2003). In this study, we screened herbicidal strains from marine microorganisms. The biological control of weeds using living microorganisms is an acceptable alternative to chemical treatment. However, whether the living bacteria can be successfully applied in fields depends on many factors, such as survival rate in soil and environmental temperature conditions (Mejri et al. 2013). Therefore, to process an appropriate formulation with a high activity after long time storage and which can be applied in a large scale is now becoming very significant. Liquid, solid, and powder substrates are widely used in the formulation of many weed biological control agents, because cell suspensions culturing for large-scale applications is impractical (Green et al. 1998). A formulated bioherbicide is a mixture of the active ingredient, a carrier or solvent that delivers the active ingredient to the target weeds, and the adjuvants that improve the survival and effectiveness of the product in adverse environmental conditions (Boyette et al. 1991; Hynes and Boyetchko 2006; Chutia 2007; Ash 2010). The ‘pesta’ formulation has its own advantages. Bacterial cells mixed in the formulation are protected from external environmental factors, and their survival and efficacy are preserved under adverse environmental conditions (Boyette et al. 1991; Shabana et al. 2003; Kinay and Yildiz 2008). The wheat-gluten matrix called ‘pesta’ has been applied to formulate granular biocontrol elements. This matrix is suitable for many dif-

CK

ferent microorganisms and ingredients (Daigle et al. 1997) and is non-toxic, cost-effective, and easy to store and use (Elzein et al. 2004). Many mycoherbicides and bacteria have been processed to ‘pesta’ formulations, such as Fusarium oxysporum (Shabana et al. 2003; Kohlschmid et al. 2009), Pseudomonas fluorescens (Daigle et al. 2002), Pseudomonas aeruginosa (Yang et al. 2014), Lasiodiplodia pseudotheobromae, and Pseudomonas aeruginosa (Adetunji and Oloke 2013). In this study, we reported that S. marcescens has bioherbicidal activity on D. sanguinalis. The potential of the strain Ha1 as a biocontrol agent was investigated. We present the isolation, identification, and physiological and biological activities of the Ha1 strain as well as the herbicidal activity of ‘pesta’ and the Ha1 metabolites.

2. Results 2.1. Bacterial isolation and screening A total of 479 bacterial strains were isolated from brine using the previously described methods. Four strains showed signs of herbicidal activity (Fig. 1). Of these four strains, the strain designated as Ha1 showed the strongest inhibitory activity against D. sanguinalis, with complete withering or 100% mortality (0.1 mL methanol with 0.2 mg mL–1 crude extracts). Thus, Ha1 was selected for the subsequent studies. The results of the arrested seed germination show that the inhibitory concentration (IC50) of the crude extracts from D. sanguinalis radicula and coleoptile were 3.332 and 2.828 mg mL–1, respectively (Table 1).

Ha17

Ha1

Ha38

Ha384

Fig. 1 Inhibition of metabolites produced by strains Ha1, Ha17, Ha38, and Ha384 on Digitaria sanguinalis growth.

Table 1 Inhibition of the culture filtrate from Ha1 on radicula and coleoptile of Digitaria sanguinalis Concentrations (mg L–1) 0 500 1 000 2 000 4 000 5 000 Data are means±SE.

Radicula (cm) 1.55±0.15 1.18±0.02 1.10±0.00 0.98±0.06 0.79±0.09 0.44±0.09

Inhibition ratio (%) 0.00 23.87 29.03 36.61 49.19 71.77

Coleoptile (cm) 2.35±0.15 2.10±0.10 1.95±0.05 1.50±0.10 1.00±0.00 0.65±0.05

Inhibition ratio (%) 0.00 10.64 17.02 36.17 57.45 72.34

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2.2. Identification and physiological characterization of the Ha1 with herbicidal activity

activity against D. sanguinalis was selected. The biomass of D. sanguinalis plants inoculated with either a bacterial cell suspension or with the formulation was measured. The cell culture suspension reduced the D. sanguinalis dry biomass by 37% compared with that of the control plants, whereas the S. marcescens Ha1 ‘pesta’ granular formulation reduced the dry biomass by 27% (Fig. 3).

Light microscopy showed that the Ha1 strain was Gram-negative and straight, obtuse, or rod-like. The strain also exhibited an outer flagellum, a diameter of 0.5 to 0.8 μm, and a length of 0.9 to 2.0 μm. The physiological and biochemical tests showed that the catalase enzyme exhibited a strong positive reaction (Table 2). The 16S rDNA gene sequences of Ha1 (comprising of 1 598 nucleotides) were obtained and submitted to GenBank (http://www.ncbi.nlm.nih.gov). The sequence showed the highest similarity (100%) to that of Serratia marcescens. A phylogenetic tree was constructed based on the 16S rDNA PCR products as well as on the coding gene sequences of the isolate and its nearest relatives (Fig. 2). The strain was identified as S. marcescens. The gene sequence was deposited in the GenBank database under accession no. KC935341.

2.4. Effects of storage temperature on viability and herbicidal activity of Ha1 formulated granules The viability of S. marcescens Ha1 in the ‘pesta’ formulation was higher at 4°C than at (25±2)°C (Table 3). The initial S. marcescens Ha1 population prior to storage was 9.23×109 colony-forming units (CFU) g–1. Eight weeks after storage, the viability of the Ha1 strain in the pesta granules at 4°C and at (25±2)°C were 4.28×109 and 3.53×109 CFU g–1, respectively. In addition, the loss of viability of the granules was considerably lower at 4°C than at (25±2)°C (Table 3). The biocontrol activity of the S. marcescens Ha1 ‘pesta’ granular formulation against D. sanguinalis growth was assessed every week during the storage at 4 and (25±2)°C under growth-chamber conditions. The results show that the inhibition of D. sanguinalis decreased with time compared with that of uninoculated plants (Fig. 4). After eight weeks storage, the living bacterium of those at 4°C and room

2.3. Evaluation of the herbicidal activity of the formulated granular S. marcescens Ha1 in the laboratory The previously discussed formulation procedure was followed by the assessment of the bioherbicide activity of the conservation of S. marcescens Ha1 ‘pesta’ granular formulation. The bacterial treatment with the highest herbicidal

Table 2 The results of physiological and biochemical tests Test code Indole test Voges-Proskauert test Methyl red test H2S test Sugar alcohol fermentation Catalase test Hydrolysis of starch test Gram Faerbung Nitrate reduction test Oxidation of glucose fermentation experiments

M

Phenomenons Rosy on the ether layer Red The medium grew red Appear black after 48 h on medium No air bubbles Air bubbles No transparent ring Red on the microscope Red Yellow

Ha1 51

bp 2 000→ 1 500→ 1 000→

Results Positive Positive Positive Positive Negative Positive Negative Negative Positive Oxidation fermentation

39

QQ465847.1 Serratia marcescens strain SB08 NR036886.1 Serratia marcescens subsp. sakuensis strain KRED

32

FJ853424.1

9

Serratia marcescens strain MH6

JX566540.1 Serratia sp. 1136

4

HF568868.1 Serratia sp. TSS

6

HM245061.1 Serratia sp. FS014

25

KC169816.1 Serratia sp. CC-SYL-A JX566600

Serratia sp. 4034

AF124038.1 Serratia marcescens CP003959.1 Serratia marcescens WW4 100

Fig. 2 Ha1 PCR products and phylogenetic tree.

strain Ha1

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0.10

83%, showed higher inhibiting activity compared with the granules stored at room temperature, whose inhibition was reduced to 17 from 70%.

a

0.09

Dry biomass (g)

0.08

b

0.07

3. Discussion

b

0.06 0.05 0.04

0.085

0.03

0.054

Biological control of weeds using specific phytopathogenic microorganisms or microbial toxins has been a widely accepted by management strategy (El-Sayed Sayed 2005; Caldwell et al. 2012). Plant pathogens can infect the leaves or the other parts of the plants so that the growth and enlongation of the weeds were inhibited, which is an interesting and promising alternative products of chemical herbicides (Mejri et al. 2010) in the coming dacades. A number of studies have shown that the Deleterious rhizobacteria (DRB) belonging to the Pseudomonas spp. are promising in decades for weed biological control (Kennedy et al. 1991; Flores-Vargas and O’Hara 2006; Kremer and Kennedy 2007; Zermane et al. 2007; Caldwell et al. 2012) and the cell suspension of DRB has been processed to an effective bioherbicide (Mejri et al. 2013). In this study, four bacterial strains named Ha1, Ha17, Ha38, and Ha384 were isolated from microorganism and the extractions of

0.063

0.02 0.01 0

Control

Cell suspension Pesta granular

Fig. 3 Efficacy of different formulations of Serratia marcescens Ha1 on the dry weight of D. sanguinalis in pots under controlled conditions. Values are the means of the combined data of two experiments. Bars represent ±SD; means designated by the same letter are not significantly different (P=0.05) according to Fisher’s least-significant difference test. The same as below.

temperature stored in granular formulation were decreased by 4.3 and 5.5 units, respectively. The granular formulation stored at 4°C, which inhibit rate was reduced to 23 from

Table 3 Viability of Serratia marcescens Ha1 in the pesta formulation after storage at different temperatures Storage temperature (°C) 4 25±2

Weeks after storage 0

1

2

3

4

5

6

7

8

9.23±0.12 a 8.05±0.22 a 6.96±0.41 a 6.10±0.18 a 5.46±0.15 a 5.03±0.18 a 4.59±0.13 a 4.40±0.21 a 4.28±0.14 a 9.23±0.11 a 7.38±0.17 b 6.24±0.11 b 5.26±0.10 b 4.75±0.07 b 4.29±0.03 b 3.98±0.10 b 3.76±0.04 b 3.53±0.09 b

Means designated with the same letters in each column are significant (P=0.05) according to LSD Duncan significance difference test. Values are means (109 colony-forming units (CFU) g–1 of formulation)±SE.

90

Control

83

80

74

70

Inhibition ration (%)

70

(25±2)°C

72

62

4°C

66

58

60

51 45

50

50 40

41 33

40

25

30

23 17

20 10 0

9

5 1

8

6 2

3

5

6 4

5

7

5 6

7

8

Time (week)

Fig. 4 Biocontrol activity of S. marcescens Ha1 pesta granular formulation against D. sanguinalis at storage different temperatures.

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the filtrate were proved to be toxic to D. sanguinalis seeds. Furthermore, the cell suspension of Ha1, which was identified as S. marcescens, also exhibited herbicidal activity, which was consistent with the results of DRB (Mejri et al. 2013) and it is the first reported evidence for the herbicidal activity of S. marcescens. Researches on these herbicidal bacteria strains have opened up a new pathway to the progress of microbial source pesticides, demonstrating that microbial pesticides is not only confined to the fungus. Because of the low survival in liquid inoculants (Albareda et al. 2008), bacteria was not suitable for the liquid formulation. In order to overcome the disadvantage of the limitation on applying in a large scale, we processed the formulated granules of strain Ha1 and analyzed the effects of temperature on the shelf life of the formulated granules. Indeed, low temperature (4–10°C) storage can slow the rate of cell division and metabolism, thus the nutrients consumption and the toxic metabolites accumulation could be reduced to a large extent, and the moisture in the carrier are protected from being leaked out, therefore favoring the long term storage of the bacterial inoculants (Van Shrevan 1970; Trivedi et al. 2005). As a consequence, the survival of Pseudomonas trivialis X33d in ‘pesta’ granules was found to be higher after a 6-mon storage at 4°C than that at (25±2)°C (Mejri et al. 2013), which proved that low temperature of storage has been a successful reserve condition for many formulated bacterial biological control agents. In addition, the effectiveness of a bioherbicide is affected by numerous factors during formulation, for example, the storage temperature and different adjuvants (Connick et al. 1996). Of them, storage temperature affects the quality of the formulation mostly and thus determines the efficacy and shelf life of the bioherbicide. For this reason, the shelf-life of the Ha1 ‘pesta’ granules was evaluated during storage at two different temperatures (4°C and (25±2)°C). According to literature, ‘pesta’ granules are considered nonviable if the concentration of the bacterial cells in the granules is less than 1×105 CFU g–1 (Daigle et al. 2002). The results of this study showed that the bacterial cell concentration exceeded 105 CFU g–1 in the ‘pesta’ granular formulation after a 2-mon storage regardless of the storage temperature. It indicated that the strain Ha1 is suitable for being processed into granules. Environmental conditions including temperature, moisture, sunlight and soil characteristics can greatly influence the efficacy of biocontrol agents, so it is important to find out if the present formulation can withstand adverse environmental conditions or not. Therefore, the assessment of the survival rate and the herbicidal activity of the stored ‘pesta’ granular formulation mixed with different adjuvants under greenhouse or field conditions must to be done as described in the study of Fusarium oxysporum, a biocontrol agent of Phelipanche ramose (Kohlschmid et al. 2009). And

the effect of this formulation on the plant growth and root architecture were to be researched in the next experiments. The mechanisms of the bioherbicides in inhibiting plant growth mainly involve the synthesis of a large amount of phytohormones, such as indole-acetic acid, and the production of various phytotoxins and cyanide (Nehl et al. 1997). So the separation and purification of herbicidal metabolites produced by Ha1 also needs to be conducted in order to explain the mechanism.

4. Conclusion The herbicidal strain Ha1, isolated from brine was identified as S. marcescens by using the methods of 16S rDNA sequence analysis and the physiological. And the viability of Ha1 was preserved after being processed to granular formulation and the herbicidal activity was improved when the granules were stored at 4°C.

5. Materials and methods 5.1. Source of strain and test weeds Ha1 was isolated from the brine of Bohai, Qinhuangdao City, Hebei Province, China. D. sanguinalis seeds, which were collected at the Agricultural University of Hebei, Baoding, China, in the previous years, were directly seeded into plastic pots with 10 cm diameters. All pots were placed in a greenhouse under natural light and with day/night temperatures of 28°C/20°C and an average relative humidity of 35%. The pots were watered daily.

5.2. Medium A PDA medium composed of 200 g potato, 20 g dextrose, 20 g agar, and 1 000 mL distilled water was used to isolate, screen, and purify the strains. An LB agar medium composed of 10 g sodium chloride, 10 g peptone, 5 g yeast extract powder, 10 g agar, and 1 000 mL distilled water and with a pH range of 7.0 to 7.5 was used to preserve the bacteria. An LB medium composed of 10 g sodium chloride, 10 g peptone, and 1 000 mL distilled water and with a pH range of 7.0 to 7.5 was used for the liquid fermentation of bacteria.

5.3. Isolation, screening, and evaluation of the herbicidal activity of the bacteria Seawater samples from Bohai (Qinhuangdao, China) were placed in the PDA medium by serial dilution and routinely cultured at 30°C for 48 h. Single colonies were picked

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and preserved in 50% (v/v) glycerol/ddH2O at –80°C until being used. The screened bacterial strains were inoculated in flasks that contained 150 mL of the LB broth. The flasks were placed in a rotary shaker at 180 r min–1 for 5 d. The fermentation broth was extracted three times with equal volumes of ethyl acetate. The ethyl acetate extracts were concentrated using rotary evaporation at 40°C under –0.1 kPa to obtain the crude toxin, which was subsequently dissolved in methanol. The resulting solution was applied on the leaves of D. sanguinalis using a hand sprayer (100 mL m–2) with a pressure of 75 kPa. Each treatment was repeated three times. The treatment with methanol was used as control. Exactly 72 h after treatment, herbicidal effects were observed according to the yellow withered leaves (dried and dead). The test of seed germination was used to assess the herbicidal activity of Ha1.

5.4. Identification of the apparent characteristics and physiological traits of Ha1 The characteristics of the colonies were observed using light microscopy (Fan et al. 2012) and the naked eye. Gram-Faerbung and catalase tests were also performed. The strain with herbicidal activity was identified by the 16S ribosomal DNA (rDNA) gene sequencing method. DNA was extracted from the pure cultures (for specific DNA extraction methods) (Ausubel et al. 2008). On the basis of the conserved bacterial 16S rDNA gene sequences, the 16S rDNA gene was amplified by PCR using the following primers: Eu 27F (5´-GAGAGTTTGATCCTGGCTCAG-3´) and 1492R (5´-CTACGGCTACCTTGTTACGA-3´) (Lane 1991). The PCR conditions were as follows: 95°C for 10 min, 94°C for 30 s, 55°C for 30 s, followed by 35 cycles at 72°C for 90 s, and a final step at 72°C for 10 min. The purified PCR products were then sequenced by Invitrogen Biotechnology Co., Ltd. (Shanghai, China). The sequencing results were submitted to GenBank for BLAST analysis. Phylogenetic analysis was performed using the BioEdit and MEGA version 4.0 software packages (Tamura et al. 2007). The neighbor-joining method was used for phylogenetic analysis (Saitou et al. 1987).

5.5. Preparation of the ‘pesta’ granular formulation Single colonies of the Ha1 strain were isolated and inoculated in 250-mL flasks containing 100 mL of the LB broth. The flasks were placed in a rotary shaker at 180 r min–1 and 30°C for 48 h. Bacterial cells were pooled via centrifugation (10 000 r min–1, 5 min) and resuspended in 0.1 mol L–1 MgSO4·7H2O (Mejri et al. 2013). The bacterial density of the suspension was 109 to 1010 CFU mL–1, as measured by

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a spectrophotometer (Nadjia et al. 2007). The ‘pesta’ granular formulation was applied as described by Daigle et al. (2002), with fewer modifications. Semolina and kaolin were separately sterilized at 171°C for 3 h. After cooling, a mixture of 400 g of semolina, 200 g of kaolin, and 400 mL of the Ha1 bacterial culture suspension of 1010 CFU mL–1 was blended in a vessel until the mixture formed a cohesive dough. Granules were prepared from this mixture using Rotary extrusion pelleting machine (ZLJ-6, Qiangwei Powder Equipment Co. Ltd. in Kunshan, Jiangsu Province, China) by granulating and extruding before air drying. The ‘pesta’ granular formulation was packed in plastic bags and stored either at 4 or (25±2)°C.

5.6. Evaluation of the herbicidal activity of formulated granular Serratia marcescens Ha1 in the laboratory The seed germination was tested to evaluate the effectiveness of the S. marcescens Ha1 ‘pesta’ formulation in reducing D. sanguinalis growth (Mejri et al. 2013). D. sanguinalis seeds were surface-sterilized with mercuric chloride for 2 min, washed with 75% alcohol for 2 min, and rinsed three times with sterilized distilled water for 5 min. The seeds were pregerminated on moist, sterile filter papers, and incubated in the dark at 30°C for 48 h. Fifteen pregerminated D. sanguinalis seeds were placed in 10 cm diameter pots, which were then filled with 200 g of sterilized vermiculite. Afterward, 3 g of the ‘pesta’ formulation or 20 mL of the 48 h-old bacterial suspension of 1010 CFU mL–1 was incorporated to the vermiculite in each pot. The pot experiment was performed weekly. The viability of Ha1 was determined in the ‘pesta’ granular formulation stored at 4 and (25±2)°C. Viable bacteria were counted by dilution plating on the LB agar medium. Three petri plates for each dilution and three replicate samples for each storage condition were maintained. Control pots did not receive any bacterial treatment. Plants were grown at 30°C in a light incubator with a 12 h/12 h light/dark photoperiod and 80% relative humidity. The plants were watered to saturation when required. The experiment was performed thrice. The results were from the pooled data of the three replications. Two weeks after inoculation, the D. sanguinalis plants from each pot were harvested. The roots were washed to eliminate adhering vermiculite. Afterward, the plants were dried at 70°C for 12 h. The dry weight of each plant was then recorded.

5.7. Effect of temperature on the shelf life of the S. marcescens Ha1 ‘pesta’ granular formulation The viabilities of the Ha1 formulated granules stored at 4 and

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(25±2)°C were observed weekly for 2 mon. Viable bacteria were counted by being suspended 1 g of the granular formulation in 9 mL of MgSO4 buffer. The mixture was shaken for 15 min and then plated on the LB agar medium after dilution. The colony-forming units (CFU g–1) were counted after 48 h of incubation at 30°C. Three replicate samples and Petri plates were maintained for each dilution. The final counts of the viable and culturable bacteria were determined from the average of three readings.

5.8. Data analysis Data of granular formulation with herbicidal activity were analyzed using the SAS software 8.2.

Acknowledgements This research was supported by the grants from the National High Technology Research and Development Program of China (2011AA10A206), the China Agriculture Research System (CARS-02) and the Science and Technology Support Program of Hebei, China (12220301D).

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