The mealy bug,
Insects exhibit symbiotic relationships with bacteria [
In the insect pest complex in agricultural ecosystem, mealy bugs (Homoptera: Pseudococcidae) have been rated as a major pest, since they play a dual role as pest and vector in field crops [
Since rapid and effective methods to control insect pests represent one of the many scientific achievements of the twentieth century, use of antibiotics for pest management is a viable option [
Since endosymbionts play a vital role in the physiology of their host, revealing the types of bacteria associated with mealy bug will give basic information, which may throw light on the management of this pest. The present study deals with the isolation and identification of endosymbionts by classical and molecular approach and their antimicrobial activity.
Adult females of
Pure culture of each bacterium for biochemical identification was obtained by streaking the individual colony on a fresh nutrient agar plate and incubated for 24 h at 35°C. Each well separated colony was subjected to the following biochemical tests.
The gram staining was performed using Hi-Media kit (Hi-Media Laboratories Pvt. Ltd., India) according to the manufacture’s protocol. The results were observed using Leica DMLB compound microscope (100x).
Motility of the isolates was tested using hanging drop method (Hi-Media). Take a clean, scratch-free glass slide. Place 2 drops of culture in the middle of the slide and place a clean cover slip over the drop and examined under microscope.
Monosaccharide (fructose), disaccharide (sucrose, lactose, and maltose), polyhydric alcohol (mannitol), and hexose (dextrose) were tested. Bromothymol was used as the indicator. Triple sugar-iron agar test (TSI) was also done according to the manufacture’s protocol (Hi-Media, Mumbai, India).
The production of tryptophanase, sufficient amount of alcohol, acetyl methyl carbinol, and citrate utilisation were noted using the indole test, methyl red (MR) test, Voges-Proskauer (VP) test, and citrate utilization test, respectively, according to the protocol of Mackie McCartney [
Exoenzymes produced were noted using urease test, O-nitrophenyl-beta-D-galactoside (ONPG) (
Pure cultures were maintained by subsequent subculture/transfer in nutrient agar medium every 15 days. Cultures were preserved by freezing at −70°C in 20% glycerol.
Total genomic DNA was extracted from bacterial cultures and treated with 100
PCR products were gel-purified and ligated into T/A cloning vector PTZ57R/T (InsTAclone PCR cloning kit, Fermentas, EU) and transformed into competent DH5
The nucleotide sequence obtained was processed to remove low quality reads and transformed into consensus sequences with Geneious Pro software version 5.6. The resulted high quality sequences were analyzed with BLASTn (NCBI;
Antibiotic susceptibility of each isolate was determined by the disk diffusion method [
A total of five bacterial strains were successfully isolated from the
Isolates Rhizo 1, Rhizo 2, and Rhizo 3 showed similar biochemical characters (Table
Phenotypic characters of bacterial isolates from mealy bug,
Isolate | Colony characters | Gram reaction | Shape | Acid production | ONPG | ||||||
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Dextrose | Lactose | Sucrose | Mannitol | Maltose | Fructose | Starch | |||||
Rhizo 1 | Circular, entirely flat | P | Rod | P | P | N | N | N | N | N | N |
Rhizo 2 | Circular, entirely flat | P | Rod | P | P | N | N | N | N | N | N |
Rhizo 3 | Circular, entirely flat | P | Rod | P | P | N | N | N | N | N | N |
Rhizo 4 | Irregular, creamy yellow, lobate | P | Cocci | P | P | P | P | P | P | P | N |
Rhizo 5 | Small, irregular, lobate, white | P | Cocci | P | P | P | P | P | P | P | N |
Isolate | Urease | IMViC | TSI | Nitrate production | MIO | Motility | Phenotypic identification | 16S rDNA identification | |||
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Indole | MR | VP | Citrate | ||||||||
Rhizo 1 | SP | N | N | N | P | Acidic slant, acidic butt | P | N | Motile |
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Rhizo 2 | SP | N | N | N | P | Acidic slant, acidic butt | P | N | Motile |
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Rhizo 3 | SP | N | N | N | P | Acidic slant, acidic butt | P | N | Motile |
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Rhizo 4 | P | N | P | N | P | Acidic slant, acidic butt | P | N | Motile |
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Rhizo 5 | P | N | N | N | P | Acidic slant, acidic butt | P | N | Nonmotile |
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P: positive, N: negative, and SP: slightly positive.
The isolate Rhizo 4 was motile, gram positive, and cocci shaped. Nutrient agar colonies were irregular, lobate, and creamy yellow in colour (Table
The isolate Rhizo 5 differed from other isolates with small, irregular, and white colonies. They were gram positive and cocci shaped and even though all other isolates were motile, this bacterium was nonmotile. Acid was produced from all sugars tested. IMViC test showed positive to citrate only (Table
The biochemical characters of all isolates were verified using Bergey’s Manual of Systematic Bacteriology Volume 2 (2005) and Bergey’s Manual of Systematic Bacteriology Volume 3 (2009). Based on morphological and biochemical characteristics (Table
Although biochemical tests revealed the preliminary identity of the isolates, for definitive identification 16S rDNA sequencing was performed.
The bacterial isolates were identified based on 16S rDNA sequencing. PCR amplification yielded ~1600 bp amplicon in all the isolates. BLAST analysis of isolates Rhizo 1, Rhizo 2, and Rhizo 3 showed 100% similarity to
Neighbor-joining (NJ) tree showing the evolutionary relationship of
Neighbor-joining (NJ) tree showing the evolutionary relationship of
The antibiotic activity against the endosymbiotic bacteria isolated from
Antibiotic activity against the endosymbiotic bacteria of
Antibiotic activity (cm) | ||||
---|---|---|---|---|
Cephalexin | Ciprofloxacin | Endrofloxaxin | Cefixime | |
Rhizo 1 | R | 3.7 | 2.2 | 3.8 |
Rhizo 2 | R | 3.4 | 2.8 | 4.0 |
Rhizo 3 | R | 3.5 | 2.7 | 3.8 |
Rhizo 4 | R | 2.5 | 2.3 | 2.8 |
Rhizo 5 | R | 2.8 | 2.7 | 3.1 |
Almost all insects have endosymbionts for their normal growth and development [
Isolates 1, 2, and 3 showed similar phenotypic characters. When compared with Bergey’s Manual of Systematic Bacteriology (2009) it was seen that the biochemical tests of three isolates showed similarity with
Classical biochemical identification at the species level was found very difficult and often confusing [
Bacteria of genus
The phenotypic characteristics of 15 probable species of
The bacterial association with arthropods has been used to develop novel control strategies of agricultural, medical, and veterinary important pests [
Antibiotic susceptibility tests showed that all the isolates were resistant to the antibiotics except cephalexin. Antibiotic sensitivity pattern of
Endosymbionts could be used as effective biocontrol agents (BCAs) and the present study will stand as a connecting link in identification and effective utilization of these endosymbionts as BCAs and also in formulating newer IPM strategy for
The authors declare that there is no conflict of interests regarding the publication of this paper.