Phylotype Analysis of Ralstonia Solanacearum Causing Bacterial wilt in Eggplants in Orissa in India

Copyright © 2019 by author(s) and International Journal of Trend in Scientific Research and Development Journal. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0) (http://creativecommons.org/licenses/ by/4.0) ABSTRACT Eggplant is prone to attack by several pests including bacteria, fungi, nematodes and insects. In this study, we have analyzed phylotype of bacterial wilt (Ralstonia solanacearum) infection in eggplant plants collected from Bhubaneswar (Orissa) in India. Bacterial wilt symptomatic five plant samples were collected from brinjal field in Bhubaneswar in 2016. The samples were macerated in sterile distilled water and grown on Kelman’s triphenyltetrazolium chloride (TZC) agar media. Total genomic DNA of the bacterium were extracted and subjected to PCR amplification using the R. solanacearum specific universal primer pair 759/760. An expected single 280 bp fragment amplified in all the samples confirmed the identity of these as Ralstonia. To reconfirmed isolate of bacterium, the amplicon was sequenced in sequencer. In NCBI blast, the nucleotide sequence was 100% similar with Ralstonia solanacearum strain RS-lpxC-DOB-1 (AB910593) and the sequence was submitted in NCBI database under Acc. No. KY393266. To determined phylotype of strain used specific multiplex PCR with phylotype specific primers (Nmult: 21F1/2, Nmult: 22InF, Nmult: 23AF, Nmult: 22RR) revealed that all the five infected samples belonged to phylotype I as a 144-bp amplicon were observed in agarose gel. On the basis of above finding concluded that the bacterial wilt infected eggplant collected from Bhubaneswar was Ralostonia solanacearum, Phylotype I.


INTRODUCTION
Brinjal or eggplant (Solanum melongena L.) is an important solanaceous crop of sub-tropics and tropics region. India produces 12.5 million ton in a year and stand second position in the total world production of eggplant (Solanum melongena) after China (FAOSTAT, 2017). In India, it is one of the most common, popular and principal vegetable crops grown throughout the country except higher altitudes. Major eggplant growing states and its percentage production share in India are West Bengal (24%), Orissa (16%), Gujarat (11%), Madhya Pradesh (9.2%) and Bihar (9%) (National Horticulture Board, India, 2015-16).
The eggplant production limits up to 86% because of bacterial wilt disease (BW) caused by Ralostonia solanacearum in India (Sabita et al., 2000). The Ralstonia solacearum ranks among the most devastating pathogen infecting solanaceous crops viz, tomato, potato, brinjal etc. (FAO). The host of bacterium is more than four hundred plant species and spread across the world (Patil et 2017). This soil born bacterium has been classified into five races and six biovars based on host and trophic traits respectively (He et al., 1983). More recently, based on geographical origin of the strains of the bacterium, it is divided in four phylotypes: Phylotype I (Asia), Phylotype II (America), Phylotype III (Africa) and Phylotype IV (Indodenia) . In India, All the three phylotype (I, II, IV) are reported to infect potato crops (Patil et al 2017) but Phylotype I is reported to infect solanaceous crops including eggplants from different states of India but not from Orissa (Ramesh et al., 2014). In this study, we are reporting phylotype of Ralstonia solacearum infecting eggplants in Orissa.

Material and methods Bacterial strains, media and growth condition
Five samples were isolated from bacterial wilt affected brinjal plants collected from three different brinjal growing regions of Bhubaneswar of Orissa in India during Kharif 2016 season. Stem pieces (8-10 cm long) of wilted brinjal plants were collected from each field washed thoroughly; air dried and brought to the laboratory for further studies. The samples were then surface disinfected with 70% ethanol, peeled, sub sampled and macerated in sterile distilled water (Fig. 1A). Macerates were streaked on Kelman's triphenyltetrazolium chloride (TZC) agar medium (Peptone, 10 g; glucose, 5 g; Casamino acid, 1 g; agar, 17 g; TZC, 50 mg L -1 ; pH 6.5) (Kelman, 1954). Plates were incubated at 28°C for up to 72 h. Bacterial colonies developing the typical

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International Journal of Trend in Scientific Research and Development (IJTSRD) @ www.ijtsrd.com eISSN: 2456-6470 Page: 2 irregular mucoid colonies were again streaked onto fresh TZC medium for further purification. Well separated typical wild type R. solanacerarum colonies were further transferred to medium modified by exclusion of TZC for multiplication of inoculums ( Fig 1B).

Results and discussion Bacterial strain collection and their identification
In this studies, bacterial wilt infected brinjal stems were collected in Kharif 2016 season from wilt affected areas of Bhubaneswar of Orissa state in India. A total of five bacterial culture were recovered from wilt affected brijal stems. On Kelman's (1954) TZC agar medium, these strains yielded typical virulent type colonies, which were cream coloured, irregularly shaped, highly fluidal with pink pigmentation in the centre (Figure 1d). These characters were consistent with R. solanacearum as described by Kelman (1954) on TZC agar medium. Total genomic DNA of all the culture was extracted and subjected to PCR amplification using the R.
solanacearum specific universal primer pair 759/760. An expected single 280-bp fragment (Opina et al., 1997) amplified in all the strains (Fig. 1C), which further confirmed the identity of these strains as R. solanacearum.

Sequence analysis
One PCR positive samples Out of five samples amplified with primer pair 759/760 was PCR clean up followed by sequencing ABI 3130 Genetic Analyzer (Applied Biosystems, USA). The sequence was blast in NCBI data base and found 100% similarity with Ralostonia solacearum strain RS-lpxC-DOB-1 (AB910593) infecting Solanum lycopersicum (Tomato) in Karnataka and the sequence was submitted in NCBI data base under Acc. No. KY393266.

Phylotype identification
Phylotype specific multiplex PCR revealed that all the five strains from Bhubaneswar belonged to phylotype I as a 144bp amplicon was observed in all the strains when Pmx-PCR products of these strains were subjected to electrophoresis on 1% agarose gel (Fig. 1D)