Abstract: Marine organisms continue to be a huge source to produce unique and potential bioactive compounds that cure deadly disease. This research investigates the herbicidal properties of bacteria isolated from surface of the coral, Junceella juncea (Pallas, 1766). Two hundred and thirty four different bacterial strains were isolated from the coral from Tuticorin coast, Gulf of Mannar region, south east coast of India. The strain WP3 was found to prove excellent herbicidal property when tested against Lemna minor. The 16S rRNA sequencing and phylogenetic identification shows that the stain WP3 was identified to fell under the genera Bacillus. The assay was carried out for crude extract and column purified extract. Mass spectrophotometry (MALDI-TOF) analysis has shown that the mass of the molecules ranged from 1148-1729 Da. Thus the marine bacteria isolated from corals are a potential source of novel bioactive agents and other natural products.
INTRODUCTION
World's oceans provide vast source of biological compounds that are highly significant for the discovery of therapeutic agents (Wright, 1998). The oceans represent the most diverse resource of life with huge dimensions and extreme variations in pressure, salinity and temperature. A unique adaptation strategy has to be acquired to sustain these extreme conditions, leading to new natural products (Faulkner, 2000). In marine environment, bacteria grow on submerged biotic and abiotic surfaces (Chellaram et al., 2012a; Dunne, 2002). In particular, a diverse community of surface attached bacteria was found on the surface of marine invertebrates (Rohwer et al., 2002; Chellaram et al., 2012b). Several factors including surface-specific interactions account for the diversity of these organisms (Rohwer et al., 2002; Taylor et al., 2005). Weeds reduce crop yield by competing for water, light, soil nutrients, space and carbon dioxide. Weeds also pose additional problems such as reducing crop quality by contaminating the commodity, interfering with harvest and serving as hosts for crop diseases. Nowadays the appropriate application of herbicides has brought about a great reduction of labour and increase in crop yield to support the world population (Anand et al., 2012). Active compounds from marine sources can be developed into herbicides to aid agriculture. Natural products minimizes environmental pollution, thus highlighting the need for bioactive compounds from marine sources. The methanol extract of salt marsh Salicornia brachiata, Sesuvium portulacastrum and Suaeda maritima were found to have herbicidal activity against Lemna minor (Anand et al., 2014). They isolated bacterial stains from the surface of sponges, seaweeds, crabs, ascidians and cephalopod eggs and reported that the bacterial broth extracts exhibited 90% inhibition against L. minor (Anand et al., 2012).
Here, our aim is to isolate and identify bacteria from sea fan and analyze its herbicidal activity. The isolated marine bacteria was identified as Bacillus kochii using 16S rRNA sequencing technique. The marine bacterium was found to possess bioactive compounds that provides herbicidal activity.
MATERIALS AND METHODS
Sample collection: Sea fan coral, Junceella juncea was collected from a depth of 5-10 m at Tuticorin coastal waters, Gulf of Mannar region, south east coast of India. A single branch of the coral was gently cut off and care was taken not to disturb the whole organism. The collected samples were then placed inside sterile ethylpolythene bags underwater and transferred to the laboratory aseptically in iceboxes.
Isolation of bacteria: The coral sample was first washed gently with sterile seawater to remove sand particles. Isolation of epibiotic bacteria was done by swabbing a small area of the coral surface with a sterile cotton swab. The swab was then directly swabbed on Zobell Marine Agar (ZMA) plates. The ZMA plates were incubated at room temperature for six days and from the fifth day on colonies of different morphotypes were isolated and repeatedly streaked on Zobell marine agar plates to obtain pure cultures. The pure cultures were then stored at 4°C in marine agar slants until further studies.
Cold-ethanol precipitation: The cold-ethanol precipitation of the culture broth was carried out following the slightly modified method of Schubert and Finn (1981). The Bacillus strain WP3 culture was centrifuged at 7500 rpm at 4°C for 15 min. To the supernatant two volumes of ice-cold ethanol was added gradually simultaneously agitating with a magnetic stirrer. When the solvent addition was complete, the culture was agitated at 4°C for at least 60 min. The culture was then placed in an ice bucket and left overnight inside a cold room (4°C). The precipitate was separated from the supernatant by centrifugation at 7000 rpm for 30 min in 4°C. The precipitate was dried in room temperature to remove the ethanol and then dissolved in 5 mL of MilliQ water. The herbicidal activity of the ethanol precipitate was carried out.
Herbicidal activity of pure compounds: Herbicidal activity was studied by using Lemna minor L. (duckweed) as test plant. Lemna minor L. is a miniature aquatic monocot, Lemna plant which consists of a central frond or mother frond with two attached daughter fronds and a filamentous root. Under normal conditions, the plant reproduces exponentially with budding of daughter fronds from pouches on the sides of the mother fronds.
Lemna plants are sensitive to bioactive compounds. These plants are used to identify anti-tumor and phyto-toxic compounds. Search for new herbicidal compounds from marine sources is limited. Scientist have been working on marine herbicidal products from recent years. The herbicidal activity of pure compounds was assayed against Lemna minor L. following a bench top bioassay. To assay the herbicidal activity, 2 mg of the pure compound was dissolved in 20 mL of suitable solvent. From this solution, 100, 50 and 10 μL was pipette into dram vials corresponding to 0.01, 0.005 and 0.001 μg mL-1, respectively in triplicates. Controls were added with appropriate solvents alone without the compound. The solvents of the control and test vials were allowed to evaporate overnight and 2 mL of E-medium (consisting of KH2PO4-680 mg, KNO3-1515 mg, Ca(NO3)2.4H2O-1180 mg, MgSO4.7H2O-492 mg, H3BO3-286 mg, MnCl2.4H2O-3.62 mg, FeCl2.6H2O-5.40 mg, ZnSO4.7H2O-0.22 mg, CuSO4.5H2O-0.22 mg, Na2MoO4.2H2O-0.12 mg and EDTA-11.2 mg in 1 L of distilled water) was added into each vial. A single healthy L. minor plant containing a rosette of three fronds was introduced into each vial and was placed in glass chamber with about 2-4 cm water at the bottom to maintain the moisture content of the chamber and was sealed with glass plate. The fronds per vial was counted daily upto 10 days and symptoms of damage to the frond such as yellowing and decaying was noted.
Mass determination: Matrix Assisted Laser Desorption/Ionization Time-Of-Flight (MALDI-TOF) spectrum of crude and 7HPLC purified active fractions were acquired on Ultraflex Bruker mass spectrometer, equipped with a nitrogen laser of wavelength 337 nm. Equal amounts of samples were mixed with the matrix solution (α-cyano-4-hydroxy cinnamic acid) saturated with 0.1% TFA and acetonitrile (1:1). Measured masses have an error of ~±3Da.
Molecular identification and phylogenetic analysis of Bacillus strain WP3: Single colony of the strain WP3 was taken from the agar plate. The strain was suspended in 50 μL of lysis solution (10 mM Tris-HCl, pH 7.5; 10 mM EDTA and 50 μL mL-1 of proteinase K). The mixture was incubated at 50°C for 15 min. Proteinase K inactivation was done at 85°C for 10 min. The mixture was later centrifuged at 15,000 rpm at 4°C for 15 min. Genomic DNA, present in the supernatant was directly used as template in PCR reaction. The PCR amplification of almost full-length 16S rRNA gene was carried out with eubacteria specific primer set 16F27N (5’-CCAGAGTTTG ATCMTGGCTCAG-3’) and 16R1525XP (5’-TTCTGCAGTCTAGAAGGAGGTGWTCCAGGC-3’) (Pidiyar et al., 2002). Ten nanogram of the genomic DNA, 1X reaction buffer (10 mM Tris-HCL, pH 8.8 at 25°C, 1.5 mM MgCl2, 50 mM KCl and 0.1% Triton X-100), 0.4 mM deoxynucleoside triphosphates (Invitrogen), 0.5 U DNA Polymerase (New England Labs, UK) was used to perform a 25 μL reaction volume PCR.
An automated Gene Amp PCR system 9700 thermal cycler was used to perform PCR under the following conditions. The amplification condition was given as follows: 94°C for 1 min (denaturation), 55°C for 1 min (annealing), 72°C for 1.30 min (elongation) at and 72°C for 10 min final elongation. The PCR product of around 1.5 Kb was run by electrophoresis with 5 μL of the PCR product on 1% agarose gel in 1X TBE buffer and stained with ethidium bromide 0.5 μg mL-1. The PCR product was precipitated by PEG-NaCl (20% PEG in 2.5 M NaCl). Precipitation was done at 37°C for 30 min. Centrifugation of reaction mixture was done again at 12,000 rpm for 30 min at room temperature. The resultant pellet was washed twice with 70% ethanol. The pellet was later dried and resuspended in 5 μL of sterile nuclease-free water. Later, one microliter (~50 ng) of purified PCR product was sequenced (Pidiyar et al., 2002). The sequence analysis was done at NCBI server (http://www.ncbi.nlm.nih.gov/BLAST). The alignment of the sequence was done using CLUSTALW programmed at European Bioinfomatics site (http://www.ebi.eic.uk/clustalw “Last Time Access on This Date 2015-03-31”). Phylogenetic tree was constructed using the MEGA Software version 3.1. The sequence of the 16S rRNA gene of the Bacillus strain WP3 was deposited in GenBank.
RESULTS
Bioactivity: Testing of Bacillus strain WP3 (Fig. 1) following bench top bioassay showed that the strain exhibits broad activity, inhibiting the growth of Lemna minor plant.
| Fig. 1: | Strain WP3 (Bacillus sp.) |
| Fig. 2(a-c): | Herbicidal activity of strain WP3 (Bacillus sp.) (a) WP3, (b) Control and (c) WP3 |
| Table 1: | Herbicidal activity of strain WP3 against Lemna minor |
| H: Hexane, EA: Ethyl acetate and M: Methanol. GH: Green and Healthy, G: Green, LY: Light yellow, Y: Yellow and D: Dead | |
The epibiotic strain might have secreted bioactive compounds (Fig. 2). The crude extract (ethanol) was also found to be active against L. minor. MALDI-TOF spectrums of the crude extract (Fig. 3) give the mass of active molecules. Mass of the crude extract molecules ranged from 1148-1729 Da (Fig. 4).
| Fig. 3: | MALDI-TOF data of crude extract of WP3 (Bacillus sp.) |
| Fig. 4: | HPLC trace of crude extract of WP3 (Bacillus sp.) |
Table 1 shows the data for strain WP3 in different concentrations against L. minor for six days. Hexane and methanol extract of Bacillus kochii inhibited the growth of L. minor at 200-500 mg mL-1 on 6th day. As the concentration increases, mortality rate increases, proving the presence of active compounds in the extract.
Molecular identification and phylogeny: The strain WP3, was identified as a Bacillus sp. engaging 16S rRNA gene sequencing method. Phylogenetic analysis based on comparative analysis of the sequenced 16S rRNA indicated that the strain was closely related to Bacillus kochii strain (Fig. 5).
| Fig. 5: | Phylogenetic tree of the strain WP3 (Bacillus sp.) |
Sequence was obtained by 16S rRNA sequencing and related sequences were obtained from BLAST. Multiple sequence alignment was done and phylogenetic tree was constructed using European Bioinformatics site (http://www.ebi.eic.uk/clustalw “Last Time Access on This Date 2015-03-31”) and tree view 1.6.6. Further purification may result in the extraction of active compounds that are novel and efficient. Marine source continue to provide humanity with potential compounds that can be developed into drug against deadly diseases. These compounds can also be tested for its activity against deadly diseases like cancer, malaria, etc.
DISCUSSION
In the present study, herbicidal activity of epibiotic strain, Bacillus kochii was studied and the strain inhibited the growth of Lemna minor plant at the concentration of 200-500 mg mL-1 Marine chemicals often possess quite novel structures which in turn lead to pronounced biological activity and novel pharmacology. The study of such chemicals therefore is a very promising endeavor (Blunt et al., 2005). Microorganisms will automatically acquire there resistance towards common antibiotics by altering their metabolism and genetic structure. These microbial compounds are most prominent source for discover and production for new drugs (Zhang et al., 2009; Yuan et al., 2010; Chellaram et al., 2013). This study showed that the isolated bacterium Bacillus kochii has excellent herbicidal activity. It is evident that Bacillus kochii constitutively produces a novel compound responsible for herbicidal activity. Further study is necessary for structure and functional group elucidation of the compound using Nuclear Magnetic Resonance (NMR) and Infra-red (IR) spectroscopy, in order to practice it for agricultural advantage.
ACKNOWLEDGMENTS
Authors deliver their gratitude to SERB-DST (Young Scientist Award, No.SR/FT/LS-23/2010) Govt. of India for financial support.