Biochemical and molecular characterization of bacterial Endosymbionts associated with Hishimonus phycitis (Hemiptera: Cicadellidae), the insect vector of sesamum phyllody phytoplasma
- Mahalingappa Bandakkanavar
- May 4
- 10 min read
Abstract
The present study, were carried out at GKVK, Bengaluru on diversity of culturable bacterial endosymbionts associated with Hishimonus phycitis. The leafhoppers (H. phycitis) were collected from sesamum crop at ZARS filed unit. The isolated seven endosymbiotic bacteria (Designated as EBHP-1 to 7). The study revealed that, all endosymbionts showed negative reaction to Indole and Voges-Proskauer test (EBHP-1 to 7). In citrate test, six endosymbionts showed positive reaction (EBHP-1, EBHP-2, EBHP-3, EBHP-4, EBHP-6 and EBHP-7) and EBHP-5, showed negative reaction. Further, in the methyl red test EBHP-2, EBHP-5, EBHP-6 and EBHP-7 showed positive and remaining three showed negative (EBHP-1, EBHP-3 and EBHP-4). Isolated DNA from the seven isolates of endosymbionts from their pure culture and subjected to PCR for amplification of gene encoding 16S rRNA using Fp1/Rp2 primers. DNA isolated from all samples yield the expected amplicon of 1.4 kb PCR product, which were sequenced. The phylogenetic analysis of the 16S rRNA gene and nucleotide identity indicated that EBHP-1, EBHP-3, EBHP-6 and EBHP-7 were closely clustered with Pseudomonas sp. and shared sequence similarity of 83-93(%). Similarly, EBHP 4 and EBHP 2 clustered with Exiguobacterium sp. and shared 64-93(%) sequence identity and EBHP-5 with Achromobacter sp. and 89-91(%) sequence similarity. These results suggest that, diverse endosymbiotic bacteria associated with the Hishimonus phycitis, which may have possible role in the transmission of sesamum phyllody.
Keywords: Sesamum phyllody (SP), Hishimonus phycitis, bacterial endosymbionts and 16S rRNA
Introduction
Sesamum (Sesamum indicum L.) is one of the most important and ancient oilseed crop grown in India and many parts of the world. Sesamum seed is a rich source of edible oil. Sesamum phyllody disease is major constraint for sesamum production in India and causes yield loss up to 100 per cent, its caused by a phytoplasma, which is vectored by leafhopper species, Orosius orientalis, Circulifer haematoceps, Neoaliturus haematoceps and Hishimonus phycitis (Distant) (Naingroo, 2014; Nabi et al., 2013) [9, 10]. Among the leafhopper sps, Hishimonus phycitis is a major transmission of phyllody. Phytoplasma, is a cell wall-less bacteria which multiply in the plant phloem tissues and insect haemolymph (Bertaccini et al., 2014) [6]. Almost all insects are associated with heritable endosymbiotic bacteria. Endosymbiotic bacteria in insects exist in two forms, primary and secondary. Primary endosymbionts are vertically transmitted from mother to offspring and they provide their hosts with specific nutritional compounds that are important for their survival and development. Insect endosymbiotic microflora have played a key role in their ecological and evolutionary success (Douglas, 2011) [7]. Several roles have been characterized for bacterial endosymbionts associated with insects including nutritional ecology by providing essential nutrients that are limited or lacking in the diet or aid in digestion and detoxification of food and detoxification of toxic plant metabolites in the host's diet (Zhou and Zhang, 2013) [14]. Bacterial endosymbionts are commonly associated with insects (Beard et al., 2002) [3, 4]. This present study was aimed to shed light on diversity of culturable bacterial endosymbionts in leafhopper, H. phycitis and their biochemical characteristics.
Materials and Method
Collection and isolation of endosymbiotic bacteria from the leafhopper (H. phycitis (distant) Adults of H. phycitis leafhopper were collected from field of UAS, GKVK, Bengaluru using a aspirator and kept for starvation in petridish for 24 hours (Fig.1). Adults were surface sterilized with 70 per cent ethanol for 1 minute followed by 0.1 per cent sodium hypochloride for 1 minute then with sterile distilled water for 3 times and sterilized adults were crushed using 1 ml PBS solution. After crushing, suspension was added into eppendorf tube and it was centrifuged at 2000 rpm for 10 minutes. One ml of supernatant was taken and added in 9 ml water blank (10-1 dilution). Again one ml aliquot taken from 10-1 dilution and added into another 9 ml water blank (10-2 dilution).Then it was serially diluted up to 10-6 dilution. All media (NA, Mac Conkey Agar) were melted and poured in sterile petriplates. After solidification, 0.1ml aliqout was taken from required dilutions and added into the plates. The aliquot was spreaded using sterile spreader. All plates were incubated in incubator at 28 oC for 1 day. After incubation, colonies (isolates) were selected from plates based on morphology (size, shape, colour) and made pure culture by streaking on media plates. All isolates were preserved in agar slants.
Biochemical characterization of isolated endosymbiotic bacteria
The endosymbiotic bacteria were subjected to basic biochemical tests, including citrate utilization test and IMVIC reaction. Citrate utilization test in Simmons Citrate Agar, changes in colour as an indicator in the media, which is from green to blue, indicates positive for this test. IMVIC reactions consist of Indole production test in tryptone broth, after adding kovac’s reagent, cherry red ring on the top layer of broth indicates the production of indole (positive). Methyl Red and Voges Proskauer tests in an MR-VP broth, for methyl red test, after adding methyl red, the production of red colour indicates the positive result and having ability to oxidize glucose. For Voges Proskauer, VP reagent 1 and 2 were added, and then pinkish red colour appeared which indicates the positive result. According to the method described by Benson (2002)
Molecular identification of bacterial endosymbionts DNA extraction and PCR amplification
The isolated bacteria were multiplied in Luria Bertani (LB) broth and bacterial genomic DNA was isolated by Sucrose buffer method. Kept isolated DNA at -20° C. The 16S rRNA gene was amplified from isolated genomic DNA of bacteria using forward primer and reverse primer. The full length bacterial 16S rRNA fragments were amplified by PCR from each representative isolate using primers, Fp1 forward primer (5’-AGAGTTTGATCCTGGCTCAG- 3’) and Rp2 reverse primer (5’-ACGGCTACCTTGTTACGACTT- 3’). The PCR was carried out in a total volume of 30µl. The 16S rRNA fragment was amplified in PCR. The PCR program for the amplification of 16S rRNA gene consisted of initial denaturation at 94 °C for 4 minutes, 32 cycles of denaturation at 94 °C for 1 minute, annealing at 58 °C for 1 minute and extension at 72 °C for 1.30 minute with a final extension at 72 °C for 10 minutes. Amplification was performed in a DNA thermal cycler. The aliquot of each PCR amplified product was electrophoresed on a 1.0 per cent agarose gel in 1X TAE buffer at 70 V for 45 minutes, stained with ethidium bromide and the PCR products were visualized with a gel documentation unit.
Phylogenetic analysis
The PCR products were sent for sequencing. DNA sequences corresponding to 16S rRNA gene, obtained from individual bacteria. Further DNA sequences were aligned using the clustlX software. The aligned DNA sequences were compared with the database of National Centre for Biotechnical Information (NCBI) for the identification of bacteria homology searches with 16S rRNA sequences in Gene Bank performed with Basic Local Alignment Search Tool (BLAST) program. Phylogenetic tree was developed using 16S rRNA sequence from the bacterial endosymbionts sample along with 12 sequences of bacterial endosymbionts strains downloaded from GenBank database (Table 1). A phylogenetic tree was constructed using the neighbour joining phylogenetic method using MEGA7 program with 1000 bootstrap replications. The nucleotide sequence identity matrixes for the bacterial endosymbionts were generated using Bio edit Sequence Alignment Editor (Vs. 5.0.9).
Results and Discussion
Isolation and biochemical characterization bacterial endosymbionts
growth and development. Loss of these microorganisms often results in abnormal development and reduces survival of the insect host. Previously, many reports described the isolation of bacteria from leafhopper and other sap sucking insects. However, the present study is the first to identify bacteria associated with leafhopper, H. phycitis, employing biochemical characterization and 16S rRNA sequence analysis.
The present study results reviled that, the total seven endosymbiotic bacteria were successfully isolated from the H. phycitis and were assigned code numbers as endosymbiotic bacteria EBHP-1 to 7 for laboratory purposes (Plate 1a &1b). Similar results were found in previous work of Vijaykumar et al. (2019) [13] and Anand et al. (2009) [2] they were isolated five different bacteria from Diamondback moth (Plutella xylostella) and eleven isolates from digestive tract of Bombyx mori respectively. Similar results were shown when five bacterial isolates (Rhizo 1, Rhizo 2, Rhizo 3, Rhizo 4, Rhizo 5) were isolated from stored tuber mealybug, Rhizoecus amorphopalli.
Biochemical characterization bacterial endosymbionts All the bacterial endosymbionts were subjected to biochemical tests, after 48 hours. Observations were recorded and presented in Table 2 and Plate 2.
We observed that the all endosymbiotic bacteria were developed cherry red colour in the tubes after adding kovac’s reagent to each tubes (positive test). This indicated that all endosymbionts showed negative reaction to Indole production test. Ravikumar et al. (2014) [11] reported that, isolates Rhizo 1, Rhizo 2, Rhizo 3 were identified as Bacillus sp. as they showed negative for indole production.
Voges-Proskauer test
After adding V-P reagent I (napthol solution) and V-P reagent II (40% potassium hydroxide) to tubes including uninoculated tube, we found that the ruby pink (red) colour developed in all endosymbiotic bacteria which indicated positive for this test. These results are in line with Anand et al. (2009) [2] obtained eleven isolates from digestive tract of Bombyx mori and labelled as isolate 1 to 11 and isolate 4, 7, 8, 9 and 11 shown positive for Voges- proskauer test.
Citrate utilization test
All endosymbiotic bacteria were given 48 hours incubation, observed that the endosymbiotic bacteria EBHP-1, EBHP-2, EBHP-3, EBHP-4. EBHP-6 and EBHP-7 tubes were changed the colour from green to blue indicated positive for this test whereas EBHP-5 showed negative reaction for this test which remains green in colour. Similar results were found with previous work of Ravikumar et al. (2014) [11] isolates Rhizo1, Rhizo 2 and Rhizo 3 were showed positive results for citrate utilization.
Methyl-red test
Methyl red indicator was added to each tubes including uninoculated tube and observed that endosymbiotic bacteria EBHP-2, EBHP-5, EBHP-6 and EBHP-7 were developed red colour throughout the tubes indicated positive for the test whereas, endosymbiotic bacteria EBHP-1, EBHP-3 and EBHP-6 were not found any red colour throughout the tubes indicated negative for the test. Similar results were found that, biochemical characters of isolate 2, 3, 4, 5, 6, and 10 shown positive result for methyl red test (Anand et al., 2009) [2] and isolates PMB 1, PMB 2, PMB 12, PMB 14, PMB 18 and PMB 23 reacted positive to methyl red test (Abinaya et al., 2019)
Molecular identification of bacterial endosymbionts
In total, seven endosymbiotic bacteria isolated from Hishimonus phycitis were identified and sequenced. The genomic DNA was isolated from the seven isolates of endosymbionts from their pure culture and subjected to PCR amplification of the 16S rRNA gene using universal primers. The thick DNA bands were visualized on agarose gel under gel documentation. All the isolated endosymbionts yield the expected amplicon of 1.4 kb size PCR product, which were sequenced, edited and compared with GenBank sequences using BLAST algorithms (Plate 3). Vijaykumar et al. (2019) [13] isolated bacterial genomic DNA and amplified using PCR with 16S rRNA primers (expected size 1000 bp). Amplified 4kb DNA fragments containing 16S-23S ribosomal DNA (rDNA) from the P-endosymbiont of 24 whiteflies from 22 different species of 2 whitefly subfamilies (Thao et al., 2004). The phylogenetic analysis and nucleotide identity revealed that four endosymbiotic bacteria (EBHP-1, EBHP-3, EBHP-6 and EBHP-7) closely clustered with Pseudomonas sp. and shared sequence similarity of 93-83 per cent. Similarly, two endosymbiotic bacteria (EBHP 4 and EBHP 2) clustered with Exiguobacterium sp. and shared 64-93 per cent sequence identity and one isolate (EBHP-5) with Achromobacter sp. and 89- 91 per cent sequence similarity (Table 3 and Fig.1). Singh et al. (2012) reported that phylogenetic analysis of 16S rDNA sequences of Cardinium, Wolbachia, Rickettsia and Arsenophonus showed that each of these bacteria form a separate cluster when compared to their respective counterparts from other parts of the world. Munson et al (1992) compared the 16s ribosomal RNA from the endosymbionts of three species of mealybugs and parsimony analysis of the sequences indicated that the mealybug endosymbionts are related and belong to the P-subdivision.
Acknowledgments
I would like to precise my sincere gratitude to Dr. H. K. Ramappa, Professor of Department of plant pathology, UAS, GKVK, Bengaluru, for the continuous support of my Ph.D. study and research. Also to Dr. Shivanna, Associate professor, Department of Agricultural Entomology, UAS, GKVK, Bengaluru, for providing laboratory facilities. This investigation is a part of the Ph.D. (Plant Pathology) degree program.
References
1. Abinaya K, Kennedy JS, Anandham R. Biochemical characterization of culturable bacterial endosymbionts of papaya mealybug, Paracoccus marginatus Williams and Granara de Willink (Hemiptera: Pseudococcidae) from papaya plants. Journal of Entomology and Zoology Studies 2019;7(5):1324-1327.
2. Anand AAP, Vennison SJ, Sankar SG, Prabhu DIG, Vasan PT, Raghuraman T, Geoffrey CJ, Vendan SE. Isolation and characterization of bacteria from the gut of Bombyx mori that degrade cellulose, xylan, pectin and starch and their impact on digestion. Journal of Insect Science 2009;10(1):107.
3. Beard CB, Cordon-Rosales C, Durvasula RV. Bacterial symbionts of the triatominae and their potential use in control of Chagas disease transmission. Annual Review of Entomology 2002;47(1):123-141.
4. Beard CB, Durvasula RV, Richards FF. Bacterial symbiosis in arthropods and the control of disease transmission. Emerging 1998;4(4):581.
5. Benson HJ. Microbiological applications. Boston, MA: McGrow Hill 2002, P170-200.
6. Bertaccini A, Duduk B, Paltrinieri S, Contaldo N. Phytoplasmas and phytoplasma diseases: a severe threat to agriculture. American Journal of Plant Sciences 2014;5:1763-1788.
7. Douglas AE. Lessons from studying insect symbioses. Cell Host and Microbes 2011;10(4):359-367.
8. Feldhaar H, Gross R. Insects as hosts for mutualistic bacteria. International Journal of Medical Microbiology 2009;299:1-8.
9. Nabi SU, Madhupriya, Rao GP. Hishimonus phycitis (Distant), a potential vector for sesame phyllody phytoplasma in India. Conference: National Symposium on Functional Strategies for Tackling Emerging Diseases of Major crops, held at IARI New Delhi 13th December, IARI New Delhi 2013.
10. Naingroo SUN. Multilocus gene characterization of sesame phyllody phytoplasma and identification of its insect vector. M.Sc. (Agri.) Thesis submitted to the to IARI India 2014, P101.
11. Ravikumar SS, Jayaprakas CA, Ragesh L, Sasidharan N, Kumar. Endosymbiotic Bacteria Associated with the Mealy Bug, Rhizoecus amorphophalli (Hemiptera: Pseudococcidae). International Scholarly Research Notices 2014;23:23-29.
12. Sacchi L, Genchi M, Clementi E, Bigliardi E, Avanzati AM, Pajoro M et al. Multiple symbiosis in the leafhopper Scaphoideus titanus (Hemiptera: Cicadellidae): Details of transovarial transmissionof Cardinium sp. and yeast-like endosymbionts. Tissue Cell 2012;40:231-242.
13. Vijaykumar W, Muthuraju R, Shivanna B, Archana K, Nalini BS. Isolation, characterization and molecular identification of culturable gut bacteria in diamondback moth, Plutella xylostella (Linnaeus). International Journal of Current Microbiology and Applied Sciences 2019; 8(02):3291-3298.
14. Zhou QSX, Zhang Y. Symbiont-mediated functions in insect hosts. Communicative and Integrative Biology 2013;6(3):e23804.
Comments