Identification of Chitin Degrading Bacterial Strains Isolated from Bulk and Rhizospheric Soil

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The use of naturally occurring chitinolytic bacteria, actinomycetes and fungi as potential supplements for insecticides pesticides have been reported in many studies 1 .Chitin, the second most abundantly found and widely distributed natural renewable resource next to cellulose in nature.It is a homopolymer of ß-1, 4-linked N-acetyl-D-glucosamine. Chitin is the main structural component of shells of crustaceans, exoskeleton of insects, fungal cell wall and protozoa.The annual worldwide turnover of chitin is around 100 billion tons 2 .Based on amino acid sequences present chitinases are the enzymes that catalyze chitin degradation and divided into Family 18 and 19 of glycosyl hydrolase 1 .Several bacteria produces chitinases to degrade chitin and utilize it as an energy source and thereby helpful in recycling these resources in soil ecosystem 2 .
A large number of chitinolytic soil bacteria have been isolated from soil 3 , shellfish waste 4 , shrimp shell-enriched soil 5 and vermicompost 6 .Phytospheres, such as rhizosphere and phylloplane, are important habitats for chitinolytic bacteria 7,8 .There is a considerable interest in chitinolytic bacteria for efficient bioconversion of chitinaceous waste based on the exploitation of chitinases.Soil bacteria are excellent sources of chitinases and could be used for catabolic conversion of chitinaceous waste into useful molecules for application in agriculture, biotechnology and medicine 9,10 .Bacteria from genera like Bacillus, Serratia, Pseudomonas, Streptomyces and Aeromonas frequently occur in soil and are potentially suitable sources of enzymes.Recycling of chitinous waste using chemical treatments is a costly process 11 .The continous use of chemicals in agriculture leads to numerous environment and public health problems.Hence, there is a need to look forward for an environmentally sound and cost effective approach.The use of chitinolytic bacteria and chitinases (exochitinase and endochitinase) can be adopted as an alternative to both, degradation of chitinous waste and as biocontrol agent.
In the present study, bulk soil, rich in chitinous waste and rhizospheric soil rich in microbes' were collected randomly for isolation, screening of chitinolytic bacteria and performed insect mortality bioassay with selected strains.

MATERIALS AND METHODS
Local market (fish and chicken) of Delhi NCR and rhizospheric regions of Ludhiana and Meerut were randomly selected for soil collection (Table 1).Soil sampling was done using the quadrat method of sampling and was processed.

Preparation of colloidal chitin
Colloidal chitin was prepared by method described by Roberts and Seltrennikoff with modification 12 .To 2gm of chitin powder 35ml of concentrated HCl was added, incubated overnight at 4°C and next day ice cold 200ml ethanol was added to the mixture, stand overnight at room temperature and centrifuge it at 10,000(g) for 30min.The mixture was filtered through fine muslin cloth with continuous washings of distilled water.Recovered colloidal chitin was stored at 4°C until use.

Screening of chitinase producing bacteria
Quadrant streak of all the isolates were carried out on MSM plate amended with 0.5% chitin to isolate the potential bacteria based on the chitinase produced.
Further, isolates were screened using different concentrations of chitin (0.5, 1.0, 1.5, 2.0 and 2.5%) in MSM plates and incubated at 30°C for three to five days.Colonies with larger clear zone size (>=10mm) were selected.The pure isolates were preserved in chitin containing nutrient broth glycerol stock at -80°C to maintain viability.

Identification of chitinolytic bacterium
The isolates were identified through their morphological and physiological properties according to Bergey's manual of systematic bacteriology 13 (Table 2).

Chitinase assay
Colorimetric method described by Setia and Suharjona was used to determine the chitinase activity with three replications 14 .The reaction mixture consists of 1ml crude enzyme, 1.5ml of 1% colloidal chitin substrate in 200mM (pH6) potassium phosphate buffer.The reaction mix was incubated at 30°C for 2 h and boiled for 10 min to stop the reaction.Then centrifuge at 8000rpm for 20 min.collected the supernatant and added 1ml of Dinitrosalicylic acid (DNS) in 1ml of supernatant boiled for 5 min and left at room temperature to cool down.Absorbance was measured at 540nm against the standard curve of N-acetylglucosamine (GlcNAc) plotted between GlcNAc concentrations and GlcNAc absorbance values.One unit of chitinase activity was defined as the amount of enzyme required to liberate 1.0mg of GlcNAc per h.

PCR amplification of genomic DNA for 16S rDNA sequencing
The total genomic DNA was isolated from the samples.Approximately (1µl DNA) 1.3/1.5Kb,16S rDNA fragment was amplified using high fidelity PCR polymerase.The PCR products were sequenced bi-directionally using 16S forward and reverse primer 5'-AGHGTBTGHTCMTGNCTCAS-3'and 5'-TRCGGYTMCCTTGTWHCGACTH-3' respectively using gradient polymerase chain The amplified sequence was analysed using Data analysis software (seq Scape-v 5.2).

Phylogeny tree construction
For the construction of phylogeny, 16S rDNA sequences was matched with reference strains sequences (Table 3) in Genebank database (http://www.ncbi.nlm.nih.gov) and was aligned using Clustal W Multiple Alignment tool in MEGAV.7 program.

Statistical analysis
The standard statistical software Graph Pad Prism was used to carry out the data analysis.
The mean and standard deviation were used to summarize the collection of data for each measurement.Two-way analysis of variance was used to evaluate the influence of independent bacterial strains.Bonferroni multiple comparision procedure was used to determine whether the data show evidence of difference between the various classes of chitinolytic bacteria.

RESULTS
A total of 28 chitinase producing bacterial strains were isolated from the soil samples collected from different sites (Fig. 1).Out of 28 strains only 12 showed clear zone (>=10mm) when incubated in different concentrations (0.5, 1.0, 1.5, 2.0 and 2.5%) containing chitin media plates were selected and labeled as (MCPB1, MCPB2, MCPB3, MCPB4, MCPB5, MCPB6, MCPB7, MSCW8, MCPB9, MSCP10, MCPB11 and MCPB12) (Fig. 2).The formation of clear zone around the colonies indicates the presence of chitinase activity, to utilize chitin as a source of carbon and nitrogen.These finally selected 12 pure isolates were subjected to identification through biochemical tests.Observed results of biochemical tests presented in tabulated form  (Table 2) and also compared with each other (i) comparing zone diameter (in mm) using different concentrations of chitin amended MSM plates (Fig. 3) (ii) and measuring chitinase activity (Uml -1 ) in chitin containing MSM broth (Fig. 4).On the basis of preliminary bioassay data two isolates (MSCW8 and MSCP10) were found to be more potent among 12 isolates and further selected for molecular identification (16S rDNA sequencing).
The biochemical characteristics inferred that MCPB1 and MCPB2 shared similar characteristics Escherichia.MSCP10 and MCPB12 were found similar to Serratia and Pseudomonas respectively.The amplicon 16S rDNA sequences of isolate MSCW8 and MSCP10 on 1% agarose gel was compared with 500bp ladder (Fig. 5).Amplified 16S rRNA sequences showed that isolate MSCW8 (Accession no.MG066581) and MSCP10 (Accession no.MG066582) were found to be most similar to Staphylococcaceae bacterium HDMd_5 and Serratia marcescens strain S308 respectively with 99% phylogenetic similarity (Fig. 6, a and b)

DISCUSSION
Total 28 isolates were recovered from chitinous rich bulk soil and agriculture land.These isolates were further screened on the basis of potential to utilize chitin.Out of 12, two efficient isolates (MSCW8 and MSCP10) were identified using 16S rDNA sequence.These isolates were found similar to Staphylococcaceae bacterium HDMd_5 and Serratia marcescens strain S308 respectively.In present study observed chitinase activity was in range 0.181Uml -1 to 1.594Uml -1 .The zone clearance diameter around colonies is in range 6mm to 29mm.This infers that these isolates produce exochitinases for the utilization of chitin.Suharjona and Satia reported highest chitinase activity at 30°C (pH7) after 4 days of incubation in Streptomyces sp.S242 (0.162Uml -1 ), Bacillus thuringiensis (0.23Uml -1 ), Serratia marcescens DSM3012 (0.556Uml -1 ) 14 22 and B. thuringiensis sub sp.krustaki 23 .Present research was employed with the aim of utilizing chitinase producers as an efficient biocontrol agent.Mubarik et al. reported chitinase from Bacillus sp. as biocontrol agent 24 .Isolate MSCP10 and MSCW8 shows effectiveness against Plutella xylostella larvae in preliminary bioassay as observed (Table 4).After 72 h of treatment larval mortality can be easily observed.Different concentrations of isolates were mixed with the diet and larvae were allowed to feed upon.As bacteria starts multiplying in the gut of larvae, it puncture the gut lining, larvae feels pain in stomach and feed less.As a result chitinase start degrading the gut lining of larvae made of chitin.From above conducted experiment we can interpret that chitinolytic bacteria as such can be used as biocontrol agent inspite of using simple spray of chitinase directly in field or in combination with delta toxin was found to be more effective 25,26 which is a long and tedious process.Application of chitin to a plant was an effective biocontrol agent for pest insects, as it attracts chitinolytic bacteria to produce chitinase 27 .From current study data it can be concluded that isolated chitinolytic bacterial strains were effective against Lepidopteran insect P. xylostella.Further confirmation can be achieved by conducting insect bioassay with recovered strains in controlled laboratory conditions and consecutive field trials.

Table 1 .
Location of collected soil sample

Table 2 .
Morphological and Biochemical characterization of selected bacterial strains

Table 3 .
Reference strains for construction of phylogeny based on 16S rDNA sequences