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Insecticidal activity of an epibiotic Bacillus kochii from Gorgonian coral, Junceella juncea (Pallas, 1766).

According to a report by Food and Agriculture Organization (FAO), 120 billion dollars were lost worldwide due to 20-40% decrease in crop yield, because of the attack from pathogenic organisms and insect pests (Zhou et al, 2010). To ensure the constant and high productivity of crops, vast amount of pesticide were applied to control the pests. This has resulted in causing serious environmental pollution and accumulation of insecticide residues. Also, inducing a wide range of pesticide resistance was done (Jiang et al., 2000). For better productivity without ill effects, biopesticides have gained increased attention and interest among those concerned with developing environmentally friendly and safe integrated crop management (Leonard et al., 2000). At present, microbial insecticides are the main part of the biopesticide industry (Shi et al., 2000 and Xie et al., 1998). The taxonomic diversity of marine organisms is large as is their biochemical and behavioral diversity, this chemical diversity was found to contain pesticide substance close to DDT and Chlordane (Kennett et al., 1990).

Microorganisms possess exceptionally rich sources of drugs, including antibiotics, immune-suppressants etc (Chellaram et al., 2011). However, these drugs have been produced from a very small range of world's microbial diversity (Chelllaram et al., 2012). Actinomycetes have been found to be a best source of novel antibiotics and other bioactive compounds (Prem Anand, et al., 2012; Okami et al., 1979, Anbuselvi, et al., 2009; Prem Anand, et al., 2013 and Chellaram et al., 2013). The yield loss of crops was estimated to be 20-30%. An additional 10% of crop is lost due to post harvest storage and transportation (Duke et al., 1993). Cotton leafworm (Spodoptera littoralis), is considered one of the most affecting insect pests attacking crops, vegetables and fruit trees all over the world (Berlinger et al., 1997). It has the capability to develop resistance to most conventional insecticides. To prevent soil and environment pollution, biological or ecological control methods have been prioritized for limiting the destructive impacts of pest populations (Canaday et al, 1995, Hokkanen et al, 1995 and Nakasm et al, 1990). Reports indicated that actinomycetes play an important role in the biological control of insect including the cotton leaf worm spodopetra liltoralis, house fly Musca domestica (Hussain et al., 2002), Culex quinquefasciatus and Drosophila melanogaster (Gadelhak et al., 2005). In this paper, we have concentrated on isolation and identification of bacteria from sea fan corals and analyzing its insecticidal activity against Sitophilus oryzae. The isolated marine bacteria was identified as Bacillus kochii using 16s rRNA sequencing technique.

MATERIALS AND METHODS

Sample Collection

The coral (sea fan) Junceella juncea was collected by SCUBA diving from 5-10m depth 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 ethyl polythene 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. 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[degrees]C in marine agar slants until further studies.

Preliminary screening of the isolates for insecticidal activity

Direct spray technique was used for preliminary insecticidal assay. 10 test insect, rice weevil was placed inside a petridish and 5ml quantity of the culture broth was sprayed over the insects using sprayer. The petridish was covered and the survivors were recorded after 24hrs. All experiments were carried out in triplicates and control was also maintained without the extract.

Insecticidal activity of pure compounds

Insecticidal activity of pure compounds was assayed by bench top assay method (Mc Laughlin et al., 1991). 1 mg of each pure compounds were dissolved in 1 ml of the solvents, and from these 100, 50 and 10 [micro]L was poured in separate petriplates in triplicates and allowed to evaporate overnight to obtain same concentration in pg/ml of compounds. Controls with solvents alone were taken in separate petriplates and allowed to evaporate overnight. Ten healthy adults of Sitophilus oryzae were introduced into each petridish and sufficient food was provided to the test organisms so that, death due to starvation is ruled out. After 24 hours, the number of dead insects was counted and percentage of mortality was noted. The efficiency of the compounds in killing the insects was determined.

Mass determination

Matrix assisted laser desorption/ ionization time-of-flight (MALDI-TOF) spectrum of crude and HPLC purified active fractions were acquired on Ultraflex Bruker mass spectrometer, equipped with a nitrogen laser of wavelength 337nm. Equal amounts of samples were mixed with the matrix solution ([alpha]-cyano-4-hydroxy cinnamic acid) saturated with 0.1% TFA and acetonitrile (1:1). Measured masses have an error of ~[+ or -] 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 [micro]l of lysis solution (10 mM Tris-HCl, pH 7.5; 10 mM EDTA and 50 [micro]l/ml of proteinase K). The mixture was incubated at 50[degrees]C for 15 minutes. ProteinaseK inactivation was done at 85[degrees]C for 10 minutes. The mixture was later centrifuged at 15,000 rpm at 4[degrees]C for 15 mins. Genomic DNA, present in the supernatant was directly used as template in PCR reaction. PCR amplification of almost full-length 16Ss rRNA gene was carried out with eubacteria specific primer set 16F27N (5'-CCAGAGTTTGATCMTGGCTCAG-3') and 16R1525XP (5'-TTCTGCAGTCTAGAAGGAGG TGWTCCAGGC-3') (Pidiyar et al, 2002). 10ng of the genomic DNA, 1X reaction buffer (10mM Tris-HCL, pH 8.8 at 25[degrees]C, 1.5 mM Mg[Cl.sub.2], 50 mM KCl and 0.1% Triton X-100), 0.4 mM deoxynucleoside triphosphates (Invitrogen), 0.5U DNA Polymerase (New England Labs, UK) was used to perform a 25ml 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[degrees]C for 1 min (denaturation), 55[degrees]C for 1 min (annealing), 72[degrees]C for 1.30 min (elongation) at and 72[degrees]C for 10 min final elongation. PCR product of around 1.5 Kb was run by electrophoresis with 5 [micro]l of the PCR product on 1% agarose gel in 1X TBE buffer and stained with ethidium bromide 0.5 [micro]l/ml. The PCR product was precipitated by PEG-NaCl (20% PEG in 2.5 M NaCl). Precipitation was done at 37[degrees]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 [micro]l of sterile nuclease-free water. Later, one microliter (~50ng) 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). 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) using direct spray technique showed that the strain exhibits broad activity against Sitophilus oryzae

Three purified extracts of Bacillus strain (Acetone, Hexane and Methanol) was also tested against Sitophilus oryzae. [LC.sub.50] of hexane, acetone and methanol extracts were found to be 151.2 [micro]g/ ml, 126 [micro]g/ml and 100 [micro]g/ml respectively (Fig.2). Percentage of mortality of the isolated extracts against the larva of Sitophilus oryzae in different concentrations was shown in the Table 1. Concentration dependent mortality was observed. Table 2 shows the variation in between groups and within groups. P value was found to be 0.003 and F-crit value was found to be 2.86608

MALDI-TOF spectrums of the crude extract (Figure 3) gives the mass of active molecules. Mass of the crude extract molecules ranged from 1225Da to 1927Da.

Molecular identification and phylogeny

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) and tree view 1.6.6. The strain WP3, was identified as a Bacillus sp. engaging 16Ss rRNA gene sequencing method. Phylogenetic analysis based on comparative analysis of the sequenced 16Ss rRNA indicated that the strain was closely related to Bacillus kochii strain (Fig. 4).

Reverse phase HPLC of the extract was carried out. Presence of active compounds was confirmed by the peak present around RT 15.0 in trace HPLC (Fig. 5). Further purification may result in the extraction of active compounds that are novel and efficient. Marine continue to provide the mankind with potential compounds that can be developed into an insecticide source.

DISCUSSION

Out of ten isolated strains tested for activist against Sitophilus oryzae, six strains showed high activity. The strain WP3 was chosen for further studies. Three extracts (hexane, acetone and methanol) were obtained and tested for its activity. LC50 of methanol extract was found to be minimum when compared to acetone and hexane extracts. This shows that the methanol extract of the strain showed efficient activity against Sitophilus oryzae. Earlier reports show that, the insecticidal compounds have been isolated form marine source such as macroalgae, sponge, coral and parts of mangrove tree (Chellaram et al, 2013). Kabaru and Gichia (2001) screened different parts of the mangrove tree Rhizophora mucronata for insecticidal activity and found that the extract of bark and pith exhibited high toxicity (Kabaru et al., 2001). 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). Anwarul Haque et al. (2013) studied the insecticidal activity of ethanolic crude extracts of marine Streptomyces sp. against larvae of Sitophilus oryzae and showed that, at a concentration of 24 mg/ml, the isolate caused 100% mortality of the larvae.

CONCLUSION

This research has reported the insecticidal activity of methanolic crude extracts of marine Bacillus sp., which shows LC50 of 100 [micro]g/ml. Further study is necessary for structure and functional group elucidation of the compound by using Nuclear Magnetic Resonance (NMR) and Infra-red (IR) spectroscopy to be used for biological advantage.

ACKNOWLEDGEMENTS

Authors express their gratitude to SERB-DST (Young Scientist Award, No.SR/FT/LS-23/ 2010) and. Govt. of India for financial support.

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C. Chellaram [1] * and A. Alex John [2]

[1] Department of Biomedical Engineering, Vel Tech Multitech, Chennai-600062. Tmailnadu India.

[2] Research Scholar, St. Peter's University, Avadi. Chennai-600062. Tamilnau. India.

(Received: 01 April 2015; accepted: 10 June 2015)

* To whom all correspondence should be addressed. E-mail: chellaramvtmt@gmail.com

Table 1. Percentage mortality and [LC.sub.50] of marine
Bacillus strain extracts against Sitophilus oryzae

Concentration             Mortality (%)
of the extracts
(pg/ml)

                  Hexane    Acetone   Methanol
                  Extract   Extract   Extract

500                83.3      86.6        90
300                76.6      73.3       83.3
200                56.6       60        66.6
100                43.3      46.6        50
50                 26.6       30        36.6
Control              0         0         0
[LC.sub.50]        151.2      126       100

Table 2. ANOVA table for insecticidal activity of Bacillus strain

Source of       SS       df      MS         F       P-     F-crit
Variation                                         value

Between     150162.758   4    37540.689   5.597   0.003   2.86608
Groups

Within      134141.927   20   6707.096
Groups

Total       284304.684   24
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Author:Chellaram, C.; John, A. Alex
Publication:Journal of Pure and Applied Microbiology
Article Type:Report
Date:Sep 1, 2015
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