V. harveyi was isolated from water samples collected from the Muttukadu Experimental Station (MES) of ICAR-CIBA, Muttukadu at Chennai. The isolates were identified by various bio-chemical tests such as arginine dihydrolase (-), lysine (+), ornithine decarboxylase (+), gelatinase (+), Voges-proskauer (-), D-glucosamine (-) (Abraham & Palaniappan, 2004; Ananda Raja et al., 2017a) and compared with the characteristics of standard type strain V. harveyi ATCC 25919. The isolates were re-confirmed further by streaking in V. harveyi selective agar (VHSA) and stored in VHSA slants at 4 °C (Harris et al., 1996).

Leaves of mangrove plant ( R. apiculata) were collected from the mangrove forest at Pichavaram in Tamil Nadu, India (Lat 11 ° 27'N; Lan 79 ° 47'E). The leaves were washed with 10 mg/L of KMnO ₄ for 10 min to remove the epiphytes, sand and other extraneous matters, then cleaned with fresh water, later shade-dried at room temperature (RT), pulverized using sterile pestle and mortar and finally stored at -20 ° C till further use. The powder (2 g) was extracted with 100 mL of ethyl acetate using Soxhlet apparatus. The extracts were neutralized to pH 7 using 0.1 N NaOH or HCl and filtered through Whatman No.1 filter paper. The extracts were dried at 42°C using a hot air oven. For cold extraction, the R. apiculata extract was prepared by mixing 1.0 g of powder with 10 mL of ethyl acetate and then shaker-incubated at 37°C at 50 rpm for 96 h. The extract was filtered through Whatman filter paper No.1, then rotary evaporated (30°C) under vacuum and stored at 4°C for further use. The pH was neutralized as described earlier, the resultant extract was liquefied with 5 mg/mL of 30% (v/v) dimethyl sulfoxide (DMSO) and used for testing antagonism against V. harveyi (Sivakumar & Kannappan, 2013).

Antibacterial activity was ascertained against V. harveyi through “agar well diffusion assay” as described by Das et al. (2005). Cells of V. harveyi (50 μL of 10 ⁸ cfu/mL of 18 h old broth culture) were inoculated into sterile Petri dishes. The LB agar (35 mL) was poured into plates and allowed to solidify at room temperature (RT) for 1 h. Two wells (6 mm) were made on the LB agar plates using a sterile steel borer. The wells were sealed at the bottom using 10 μL of 1% soft sterile agar and filled with 200 μL of R. apiculata leaves extract. The plates were incubated at 37°C for 48 h and zones of inhibitions of bacteria around the well were measured (Das et al., 2015) excluding the well. Antimicrobial activity of this crude extract was determined by dissolving in 30% Dimethyl Sulfoxide (DMSO) at various concentrations. DMSO was used as negative control. Similarly, the extract obtained through “cold extraction” was also tested. The inoculated plates were incubated at 37°C for 24 h and zones of inhibitions were measured.

The minimum inhibitory concentrations (MIC) for the R. apiculata leaves extract was evaluated as described by Islam et al. (2008). Dilution methods were used to determine the MIC of R. apiculata extract. In dilution tests, V. harveyi was tested for their ability to produce visible growth on a series of LB agar plates. Various concentrations of extracts (5.0 to 50 μg) were examined and lowest concentration of the extract which inhibited the visible growth of V. harveyi was known as the MIC. The plates were incubated at 37°C for 24 h and 20 μL of V. harveyi (1.8 OD or 2.19× 10 ⁷ cfu/mL) was tested for the MIC on LB agar medium. The MBC was evaluated as the lowest concentration of a crude plant extract required to kill 99.9% of 20 μL of V. harveyi (1.8 OD or 2.19 × 10 ⁷ cfu/mL).

R. apiculata extract at 300 μg/ mL was added to 100 mL of LB medium. A volume of 500 μL of active 24 h old V. harveyi (1.8 OD) was inoculated into LB broth and incubated at 37°C in 100 rpm for 5 days under shaking condition. Every day, 3 mL of V. harveyi inoculum was taken out and the growth of V. harveyi was measured at OD 600 nm. Various virulence factors such as proteolytic, lipolytic, phospholipase, thermonuclease activities, crude bacteriocin production, exopolysaccharide (EPS) and proteases produced by V. harveyi with growth were estimated. Salt aggregation test (SAT) were carried out for cell surface hydrophobicity and cell adhesion was examined by bacterial adhesion to hydrocarbons test (BATH) (Soto-Rodriguez et al., 2012). Each test was performed in triplicate and values were expressed in average along with standard deviation.

Luciferase produced by V. harveyi was tested using the luciferase assay kit (LUC1, Technical Bulletin MB-260, Sigma, USA) and read by a luminometer (Victor TM X3, Perkin Elmer, USA). V. harveyi cells were harvested by centrifugation at 10,000 rpm for 5 min. The pellet was re-suspended in 333 μL of 1X cell lysis buffer per mL of V. harveyi and incubated for 10 min at 25°. The suspension was centrifuged at 12,000 rpm for 1 min at 4°. The supernatant was removed and stored in ice. Luciferase substrate (lyophilized, luciferase assay substrate was suspended in luciferase assay buffer) as cell lysate containing luciferase was equilibrated to 25° before use. Cell lysate (20 μL) was added to 100 μL of the luciferase substrate and mixed well. Readings were recorded in 10 seconds for light emission by the luminometer (Victor X3- Perkin Elmer) and expressed as counts per second (CPS, i.e., photons per second). The light intensity was nearly constant for 20 seconds. The LB broth medium and 1X lysis buffer were used as negative control for luciferase assay. For the estimation of luminescence, V. harveyi cells were harvested by centrifugation at 10,000 rpm for 5 min and its spent culture medium was used (Kannappan et al., 2013) and estimated by luminometer.

The shade dried leaves of R. apiculata powder was subjected to FT-IR BRUKER IFS 66 model spectrometer (Fig. 2) in the region 4000-400 cm ^-1 by employing standard KBr pellet technique (D'Souza et al., 2008).

Gas chromatography-mass spectrometry (GC-MS) analysis was performed by using Agilent GC-MS-5975C with the Triple-Axis Detector equipped with an autosampler. The GC column used was fused with silica capillary column (length 30 m × diameter 0.25 mm × film thickness 0.25 mm) with helium at 1.51 mL for 1 min as a carrier gas. The mass spectrometer was operated in the electron impact (EI) mode at 70 eV in the scan range of 40-700 m/z. The split ratio was adjusted to 1:10 and injection volume was 1 μL. The injector temperature was 250°C; the oven temperature was kept at 70°C for 3 min, raised to 250°C at 14°C/min (total run time 34 min). The temperature of the transfer line and of the ion source was set to a value of 230°C and the interface temperature at 240°C, full mass data was recorded between 50-400 Dalton/s and scan speed 2000. Mass start time was at 5 min and end time at 35 min. Peak identification of crude R. apiculata extract was performed by comparison with retention times of standards and the mass spectra obtained was compared with those available in the NIST libraries (NIST 11-Mass Spectral Library 2011 version) with an acceptance criterion of a match above a critical factor of 80% (Musharraf et al., 2012).

Plastic tubs were washed with 10 mg/L of KMnO ₄ solution (w/v) for 10 min and filled with 20 L of low saline water at 20 Practical Salinity Units (PSU). Disease-free (Ananda Raja et al., 2017b) postlarvae (PL 10) of P. monodon, obtained from a shrimp hatchery were acclimatized at 20 PSU for 5 days under laboratory conditions at 37 ± 1°C with continuous aeration. The average body weight of PL ranged from 17 to 18 ± 0.2 mg and stocked at 1000 numbers per tubs. The first control tub was inoculated with V. harveyi (10 mL of 1.8 OD) alone. The second tub was considered as treatment and inoculated with V. harveyi 2 gm/10L of crude R. apiculata extract. The third tub was considered as control where crude R. apiculata extract was added at 200 μg /mL alone with PL. The fourth tub was a control for PL, where neither V. harveyi nor extract was added. Aeration was provided for each container ensuring that oxygen level did not go above 4 ppm. The PLs were fed twice a day and the feed requirement per day was 15% of their body weight. All the experimental tubs were covered with plastic lid at the top to avoid external contamination. The water temperature, salinity and pH were recorded every 5 days. Experiments were carried out in triplicates. The mortality of PL was counted daily. No water exchange was given for the entire containers till 30 days. The total heterotrophic and V. harveyi counts were enumerated using LB and V. harveyi selective agar media under spread plate method (Traifalgar et al., 2009; Biswas et al., 2012).

The data were analyzed and expressed as means along with the standard deviation. Analysis of variance (ANOVA) (SPSS, ver. 16.0) was carried out to assess the treatments ( p‹0.05). Cumulative percentage mortality was calculated as CPM = [cumulative frequency / total number of observations (n)] × 100.

Crude extracts of 200, 250, 300, 350 and 400 μg/mL showed 6, 8, 10, 12 and 14 mm inhibitory zones respectively (excluding the well size 8 mm). As a positive control, 10 μL of oxytetracycline (250 mg/25 mL) showed a zone of inhibition of 23 mm whereas the DMSO negative control had no effect. Cold extraction of the crude extracts showed 5, 6, 7, 8, and 9 mm inhibitory zones respectively. The MIC for the crude extract against V. harveyi was 6 mm at 200 μg/mL and MBC for V. harveyi was 12 mm at 300 μg/mL (Fig.1).

The crude R. apiculata extract reduced the growth of V. harveyi (OD) from the fifth day. The highest OD difference compared to control was observed on the 15 ^th day (0.402) and the lowest on the 10 ^th day (0.041). The production of luminescence was decreased to 31, 44, 34, 47, 35 and 51 CPS at days 5, 10, 15, 20, 25 and 30, respectively compared to control (Table 1). The maximal reduction on luminescence was detected on the 30 ^th day (51 CPS) and the minimal reduction on the 5 ^th day (31 CPS). Production of intracellular luciferase was decreased during the study. The maximal decrease was observed on the 30 ^th day (45 CPS) and a minimal decrease was observed on the 10 ^th day (15 CPS). The maximum decrease of bacteriocin production (OD) was observed on the 20 ^th and 30 ^th days (0.385 and 0.382) and the minimum (0.18) was observed on the 15 ^th day. The maximal decrease of protease occurred on the 30 ^th day. The R. apiculata extract treatment was associated with a weak level (+) of phospholipase production by V. harveyi as compared with very high phospholipase (+++) production by the control for all 30 days (Table 1). In the SAT test, V. harveyi revealed strong hydrophobic activity for the 5 ^th to 30 ^th days in the control whereas the treatment showed moderate to weak hydrophobic activities.

The FTIR spectrum of dried powder of R. apiculata leaves (Fig. 2) and functional groups identified were compared with the FTIR standard library data. FTIR spectrum showed the presence of significant functional groups such as aromatics, alkanes, alcohol, carboxylic acids, esters, ethers, aliphatic amines and alkyl halides (Table 2).

GC-MS analysis on the crude ethyl acetate extract of R. apiculata revealed a mixture of volatile compounds. Fatty acid methyl esters were investigated quantitatively by GC-MS in multiple reactions monitoring mode and thus allowing for a better signal resolution without a preliminary fractionation of the extract. A total of 21 peaks were observed with retention times. The main chemical-constituent reported was squalene (69.2 peak area %), tocopherol (6.49%), vitamin E (6.34%), hexadecanoic acid (5.01%), octadecatrienoic acid (3.19%) and bicyclo heptane (2.39%). In the present study, the biological activity of the leaves extract of R. apiculata on V. harveyi may be due to the presence of these chemical constituents (Table 3). Chemical constituents were identified using spectrum database NIST 11 software installed in GC-MS.

Crude extracts obtained from both cold & hot extraction processes showed a significant inhibition on V. harveyi. The hot extract was greater than cold extraction in terms of inhibition. Correspondingly, Gurudeeban et al. (2013) have reported antimicrobial activity of R. mucronata leaves extracts. The chemical constituents of R. apiculata were screened and various phytochemicals such as 2-(2-ethoxyethoxy) ethanol (26.45%) and Kaur-16-ene (3.37%), benzophenone (16.09%) and 2-(2-ethoxyethoxy) ethanol (7.82%) constituents were found (Abidin et al., 2013). Manilal et al. (2010 ) have also reported that methanol extract of R. apiculata exhibited antimicrobial activity against shrimp-borne pathogens such as V. harveyi, V. vulnificus, V. alkaligens, V. alginolyticus and V. parahaemolyticus. It has again been confirmed in multidrug-resistant V. harveyi and V. cambellii by Ravikumar et al. (2010) with leaf extract of R. mucronata. Present study revealed that the solvent also play vital role in the extraction of phytochemical, the ethyl acetate extracts of leaves of R. apiculata were screened against V. harveyi. Similarly, Annapoorani et al. (2013) have reported in leaf extracts of R. apiculata against human pathogens such as Pseudomonas aeruginosa, Enterobacter spp and Staphylococcus aureus. The present work is also in agreement with Annapoorani et al. (2013) and inhibition could be due to the phytochemicals of phenolic group, alkaloids, steroids, triterpenes, flavonoids, catechin, tannin and anthro-quinone etc.

The decrease of virulence was also concordant with previous reports. Methanol extract of R. apiculata and R. mucronata (1 mg/mL) showed the virulence factors reduction of protease, pyocyanin pigments and biofilm produced by P. aeruginosa (Musthafa et al., 2013). R. apiculata extract treatment made V. harveyi strongly hydrophobic as BATH difference was 68.9 and 43.9. R. apiculata extract reduced 31 to 44 CPS of luminescence as compared to control (157 to 190 CPS). The bark extract obtained from R. annamalayana (at 1.0 mg/mL) decreased the bioluminescence produced by V. harveyi MTCC 3438 and also decreased its growth to 99% (Gao & Xiao, 2012). Present study agrees with the presence of similar kind of compound pattern also reported earlier by Abidin et al. (2013) and Satyavani et al. (2015), in the extracts of leaves of R. apiculata were reported to contain 18 phyto-compounds, major compounds 1-adamantly–p-me-thylbenzalimine, clivorin, 4-butyl pyridine, 1-oxide, acetamide and p-aminodiethylaniline, also the R. apiculata leaves were reported to contain 2-(2- ethoxy-ethoxy) ethanol (26.45%) and Kaur-16-ene (3.37%) (Selvaraj et al., 2014), whereas the bark of R.apiculata contains phenolic compounds like lyoniresinol-3α- O-β-arabinopyranoside,lyoniresinol-3α- O-β-rhamnoside, afzelechin-3-rhamnosideandbutylated hydroxy anisole which exhibited antioxidant activities (Halim et al., 2013). Hong et al. (2011) also reported essential oils, higher alkanes, acids, alcohol and esters from R. apiculata. The cumulative percentage mortality decrease in postlarvae caused by V. harveyi was found to be 10.6% on treating with R. apiculata extract till 30 days. The crude extract obtained from the bark of R. apiculata was found to contain tannin that show antimicrobial activity (Shamsuddin et al., 2013) and proved to be non-lethal to brine shrimp. But Sivakumar et al. (2014) have reported that when Ulva fasciata extract was tested on V. harveyi during P. monodon larviculture, the decrease on cumulative percentage of mortality on postlarvae caused by V. harveyi was found to be 32.4%. Methanol extract from R. mucronata also showed inhibition against V. harveyi (Ramesh et al., 2014). When shrimps were fed with R. apiculata leaves during shrimp grow–out practices, the survival and biomass was increased significantly (Nga et al., 2006). The values observed from the bio-assay of R. apiculata extract against the V. harveyi during P. monodon larviculture revealed significant differences ( p ‹ 0.05) between the R. apiculata extract treated V. harveyi infected cultures and control.

Our results indicate that the ethyl acetate extracts of R. apiculata inhibited growth and modulated virulence factors produced by V. harveyi. This extract also controlled the mortality caused by V. harveyi during shrimp larviculture. Based on this study, R. apiculata extract could be used as an alternative bio-product for aquaculture practices.