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Tahseen R. Hashmi1,2*, D. Dey1,Salam Rita Devi1, Ajit Varma2 and Ram Prasad2
1Division of Entomology, Indian Agricultural Research Institute, New Delhi- 110012, India.2Amity Institute of Microbial Technology, Amity University, Uttar Pradesh- 201313, India. Corresponding Author E-mail: firstname.lastname@example.org
ABSTRACT: Okra (Abelmoschus esculentus L.), is an important crop cultivated in Brazil, southern USA and India for its long, numerous seeded pods, used as a vegetable. A study was carried out to spot the dispersal frequency of bacterial endosymbionts and the genetic group of Bemisia tabaci feeding on okra plants, collected from Indian Agricultural Research Institute, New Delhi, India. The B. tabaci samples were examined based on mitochondrial cytochrome oxidase I sequences and settle down along with Asia II 1 population. Dispersal frequency of seven known endosymbionts namely Portiera, Rickettsia, Wolbachia, Cardinium, Fritschea, Hamiltonella and Arsenophonus were documented. The primary endosymbiont, Portiera aleyrodidarum was present in all the studied samples and a disparity was noted in the dispersal frequency of secondary endosymbionts. The statistics of irregular dispersal of secondary endosymbionts and the genetic group of B. tabaci delivers the elementary data of this notorious pest for advance studies on the control measures of this insect pest over okra plantation.
KEYWORDS: Bemisia tabaci; genetic group; vegetable; endosymbiontsDownload this article as:
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Hashmi T. R, Dey D, Devi S. R, Varma A, Prasad R. Records on Associated Endosymbionts and Genetic Group of Bemisia tabaci (Gennadius) Feeding on Okra. Biosci Biotech Res Asia 2016;13(4).
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Hashmi T. R, Dey D, Devi S. R, Varma A, Prasad R. Records on Associated Endosymbionts and Genetic Group of Bemisia tabaci (Gennadius) Feeding on Okra. Biosci Biotech Res Asia 2016;13(4). Available from: http://www.biotech-asia.org/?p=17439
India has extensive past of vegetarianism and the core vegetable crops are brinjal, chili tomato, okra, cabbage and cauliflower etc.1, which struggles from numerous insect pests. Amongst the insect pests, sweet potato whitefly, Bemisia tabaci (Gennadius) described as pest of tobacco in 18892 is a foremost threat worldwide nourishing on vegetables, fruit crops3 and pulses from 86 botanical families4. Adults and nymphs are destructive stages and usually found resting underside of the leaves. Other than sap sucking5 and excreting honey dew6, whitefly have a role in transmission of more than 115 types of virus7 to the commercial crops amongst which 90% belong to Begomovirus genus8.
Bemisia tabaci (Gennadius) is globally known as economically vital insect pest and is polyphagous in nature. It is dispersed throughout the northern and western regions of the Indian subcontinent, and has freshly arisen as a very serious pest in okra plantation and production. Bemisia tabaci is a phloem feeder as we know that plant phloem is highly enriched with carbohydrate content and due to which it lacks essential amino acids required for the growth of the pest. Microbial community be inherent in the pest recompense the insufficiency of the scarce amino acids and nutritional content9. The only noted primary endosymbiont of whitefly is Portiera aleyrodidarum10 (Baumann, 2005), whereas the secondary endosymbionts have a number of bacteria like Wolbachia (Rickettsiales)11, Arsenophonus (Enterobacteriales)12, Cardinium (Bacteroidetes)13, Rickettsia (Rickettsiales)14, Hamiltonella (Enterobacteriales)11 and Fritschea (Chlamydiales)15.
Secondary endosymbionts have been labelled to have plentiful impression on the insects, such as heat tolerance16, resistivity to parasitoids17, skill of virus transmission18, and vulnerable to insecticides19-20. Invasion of rickettsia is stated to have enhancement in fitness substantially and female biasness in the host population21. The symbionts act as both mutualist and reproductive manipulator for the host insect, with understandable positive impact on host population increases as well as the spread of symbiont in fields.
Present study, will reconnoiter the genetic group and the dispersal frequency of endosymbionts residing in B. tabaci samples feeding okra plantation in New Delhi, India. Study will deliver elementary evidences on the dispersal frequency of endosymbionts and genetic group in this region on okra plantation and helps as a supportive data for the control measure of this pest over okra crops.
Material and Methods
Sampling and DNA Extraction
Samples of B. tabaci used in the existing examination were collected from arenas of Indian Agricultural Research Institute, New Delhi, India. Samples were kept in eppendorf tubes comprising 100% ethanol and stored at -200C till handled for genomic DNA isolation. Total of 60 individuals were handled as samples. Individually flies were cleaned twofold with sterile distilled water and whole genomic DNA was take out through DNASure Tissue Mini Kit (Nucleo- pore, Genetix) as per manufacturer’s protocol. The extracted genomic DNA of each replicate was kept at -200C.
Identification of B. tabaci Genetic Group
Molecular interpretation of B. tabaci for approval of the genetic group was impelled based on mitochondrial cytochrome oxidase I (mtCOI) sequences after PCR reaction with universal primers (Table 1). The PCR program for the reaction is specified in Table 2. The products were examined in 1.0% agarose gel comprising ethidium bromide under UV illumination after a passage of 45 minute at 80 V. Through the predicted band size (Table 1) on the gels, the products (20 µl) were sent for outsource sequencing. Records for sequences were explored using the BLAST algorithm in NCBI Gene Bank, and were aligned using BioEdit version 7.2.5 (Thompson et al., 1994). Distance was calculated using the Kimura 2-parameter model of MEGA 6.
Table 1: Primers used in PCR detection of endosymbionts and genetic group.
|Targeted gene||Primer’s Sequence (5’-› 3’)||Annealing temp. (0C)/ Product size (bp)||Reference|
Table 2: PCR programs for the detectionof prevalence of Primary and Secondary endosymbionts in B. tabaci.
|Endosymbionts||Pre- denaturation||Denaturation||Cycling conditions|
|Portiera||94 0C (4 Min)||94 0C (30 s)||56 0C (2 Min)||72 0C (2 Min)||35|
|Hamiltonella||94 0C (4 Min)||94 0C (30 s)||52 0C (2 Min)||72 0C (2 Min)||35|
|Wolbachia||94 0C (4 Min)||94 0C (30 s)||55 0C (2 Min)||72 0C (2 Min)||35|
|Arsenophonus||94 0C (4 Min)||94 0C (30 s)||56 0C (2 Min)||72 0C (2 Min)||35|
|Cardinium||94 0C (4 Min)||94 0C (30 s)||52 0C (2 Min)||72 0C (2 Min)||35|
|Rickettsia||94 0C (4 Min)||94 0C (30 s)||58 0C (2 Min)||72 0C (2 Min)||35|
|94 0C (1 Min)||94 0C (1 Min)||55 0C (1 Min)||72 0C (1 Min)||35|
Screening of Endosymbionts
All the samples were checked autonomously for the incidence of endosymbiotic bacteria using specific primers amplifying the 16S rRNA gene for Portiera, Cardinium, Rickettsia, Wolbachia and Hamiltonella, and the 23S rRNA gene for Arsenophonus and Fritschea (Table 1). PCR reaction mixture’s concluding volume of 25µl, embraces of 12.5 µl Thermo Scientific maxima hot start PCR master mix, 8.5 µl molecular grade water, 1 µl of each forward and reverse primers and 2 µl genomic DNA. The PCR program for the endosymbionts is itemized in Table 2. The products were visualized in 1.0% agarose gel containing ethidium bromide under UV illumination after a migration of 45 minute at 80 V. With the expected band size on the gels, the products were used for outsource sequencing (Table 1). The gained sequences were allied to the available sequences in the databank using BLAST algorithm in NCBI.
The population of B. tabaci collected from okra plantation was evaluated with the reference sequences from NCBI and the phylogenetic analysis confirms, the specimens from okra belongs to Asia I genetic group (Fig. 1).
Figure 1: (a) Representing the phylogenetic status of B. tabaci collected from okra plantation; (b) Magnified tree
The outcomes of the present study update the circulation frequency of seven known endosymbionts in the studied flies from okra plantation and revealed a miscellaneous spreading array. All the individuals were positive with the invasion of Portiera (Primary endosymbiont) that correspondingly measured as the positive control for the class extraction of DNA. Figure 2 is the graphical exhibition of the dispersal frequency of secondary endosymbionts in the studied B. tabaci from okra plantation. Excluding Fritschea and Hamiltonella, individuals were found verminous with rest known secondary endosymbionts unevenly. The okra collected population were found infected with Arsenophonus (30%), Rickettsia (26.66%), Wolbachia (3.33%) and Cardinium (56.66%).
Figure 2: Dispersal frequency of endosymbionts of B. tabaci on okra plantation.
In the current study, samples were collected from grounds of Indian Agricultural Research Institute, New Delhi. This is a basic report on the genetic groups and their associated endosymbiotic microbiota investigated on one of the important host plant of B. tabaci. The examination description reveals that the specimens collected from okra belongs to most prevalent genetic group in the region i.e. Asia II 1 genetic group. The study ropes with the earlier conclusions that the range of genetic group in north and north-west India is restricted to Asia II 1, and Asia I with exceptional presence of Asia II 7 in Delhi and the occurrence of MEAM1 in some pockets of Gujrat22-24.
For the existence, passage and evolution of B. tabaci, the bacterial endosymbionts show a significant role12. The study designed on the accompanying endosymbionts of B. tabaci has been completed by many of researchers around the globe25-28 but a very limited work from India has been reported23-24, 29-30. Therefore, this study was carried out to give some extension in the evidence of associated endosymbionts of B. tabaci feeding on okra plantation in New Delhi, India.
The present study also discloses the percentage dispersal frequency of secondary endosymbionts in the flies feeding on okra plantation. Results disclosed the 100% presence of primary endosymbionts, Portiera and a dissimilarity was detected in the percentage circulation of secondary endosymbionts on the studied host plant. The flies feeding on okra belongs to Asia II 1 genetic group and it harbors Arsenophonus (30%), Rickettsia (26.66%), Wolbachia (3.33%) and Cardinium (56.66%), chains the verdict earlier reported on the host belongs to family Solanaceae and cotton22-24,29. Thus, outputs approve the association between the endosymbiotic bacterial groups and the genetic groups of B. tabaci and come to a contract with earlier works25-26,31.
The current study was emphasized in the direction of recording of the endosymbiont range associated with B. tabaci on okra plantation in New Delhi, India. The consequences stipulate, there is a lacuna present in the information of dispersal of secondary endosymbionts with respect to the host plants and genetic groups; and proposes an obligation for progressive assessments on the host wise frequency circulation of secondary endosymbionts and its term with several genetic groups of B. tabaci. A progressive and relative examination to disclose the facts regarding the role of these endosymbionts and the origin of uneven circulation, their efforts if any in the polyphagus nature of this insect pest is required for working on the control measure of this devastating insect pest.
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