Characterization and Confirmation of Papaya ringspot virus-W Strain Infecting Trichosanthese cucumerina at Tamil Nadu, India

Test seedlings rose in pots containing mixture of soil, sand and farm yard manure. Sap was prepared from infected leaves by grinding in a pre-chilled mortar and pestle with 1M sodium phosphate buffer (pH 7.2) at 1 ml/g of leaf. The sap was rub-inoculated on the 3-leaf stage seedlings, previously dusted with 600 mesh carborandum. After 5 min the excess sap was washed off with distilled water and plants were kept under insect proof cages for symptom expression.


Introduction
The Family cucurbitaceae comprises approximately 80-genera and over 800 species of plants. Trichosanthese cucumerina L., locally known as snake gourd or serpent gourd is an important vegetable crop in this family which is probably of native of India. It is cultivated in subtropical and tropical areas [1].
In the year 2009, the mosaic and leaf blistering symptoms observed in the Thondamuthur area and orchard, TNAU, Coimbatore, India. The samples were collected and studied for the virus identification. Biological, physical, serological and molecular behavior of the virus isolated was studied critically.

Symptomatology, virus culture and its maintenance
Leaves of snake gourd showing symptoms of mosaic, blistering vein thickening and leaf distortion were collected from the orchard, Tamil Nadu Agricultural University (TNAU), Coimbatore and Farmer's field in Thodamuthur, Coimbatore, and Tamil Nadu in 2008. These infected leaves were used as the source of inoculums.
Test seedlings rose in pots containing mixture of soil, sand and farm yard manure. Sap was prepared from infected leaves by grinding in a pre-chilled mortar and pestle with 1M sodium phosphate buffer (pH 7.2) at 1 ml/g of leaf. The sap was rub-inoculated on the 3-leaf stage seedlings, previously dusted with 600 mesh carborandum. After 5 min the excess sap was washed off with distilled water and plants were kept under insect proof cages for symptom expression.

Host range
The virus isolate maintained separately on snake gourd plants were sap inoculated to seedlings of different test plants pertaining to six families were used for host range studies.

Electron microscopy
One hundred mg of leaf tissue from artificially inoculated plants was macerated with 0.05 M sodium phosphate buffer in the ratio of 1:1 (w/v). The virus extract was centrifuged at 12,000 rpm for 5 min and the supernatant was collected. One drop of this supernatant was placed on the surface of commercially available formvar coated copper grids. The grids were allowed to dry for 5 min, washed gently with double distilled water and stained with 2% uranyl acetate. There after the excess stain was removed and the mounted grids were examined in a JEO JEM-1011 transmission electron microscope.

Physical properties of the virus isolates
Dilution end point (DEP): Serial dilution from infected leaves was prepared from 10 -1 -10 -8 by adding distilled water to the standard extract. The undiluted sap served as the control. Test plants were inoculated with mentioned diluted sap.

Thermal inactivation point (TIP):
The TIP was determined by subjecting 2 ml of the sap to temperatures viz., 35, 40, 45, 50, 55, 60 and 65°C for 10 min in a water bath. Freshly extracted sap that was not subjected to any temperature regime served as control.
Longevity in vitro (LIV): Sap from the infected leaves was divided into aliquots of 2 ml in test tubes and stored at room temperature (28 ± 2°C). At 6, 12, 18, 24, 48 h interval the sap was inoculated on seedlings of the assay host. Freshly extracted sap was used as control.

Serology
Source of antisera: Antibodies for PRSV, CMV and TSV and their positive samples were provided by DSMZ, Braunschweing, Germany.

Enzyme linked immunosorbant assay (ELISA): DAS-ELISA
was performed for detection of the virus isolates following the manufacturer's instruction (DSMZ Gmbh, Braunschweig, Germany). Purified IgG was diluted in coating buffer (1:1000) and 200 μl was added to each well of a microtiter plate. The plates were incubated at 37°C for 3 h and thereafter plates were washed with phosphate buffered saline-Tween (PBS-T), soaked for a few minutes and washing was repeated twice. Plates were tapped upside down on tissue paper. Aliquots of 200 μl each of the test sample extracted in sample extraction buffer was added to wells in duplicate. Buffer without sample served as control and plates were incubated at 4°C overnight. The plates were washed as earlier and 200 μl of the anti-virus conjugate (1:500) was added to each well and incubated at 37°C for 2 h. later the plates were washed three times.
Finally 200 μl of freshly prepared substrate (10 mg of ρ-nitro phenyl phosphate dissolved in 10 ml of freshly prepared substrate buffer) was added to each well and incubated in dark at room temperature for 45 min or as long as necessary to obtain clear reactions. Spectrometric measurement of absorbance was read at 405 nm (EL 800, BIO-TEK Instrument Inc., USA) and the reaction was later arrested by adding 50 μl of 3M NaOH.

Molecular characterization of the virus
The coat protein gene of the viral genome was amplified by reverse transcription polymerase chain reaction (RT-PCR) for confirming the virus. Total RNA was extracted from infected and healthy leaf tissues with GeneiTM plant RNA isolation kit (Bangalore Genei Pvt. Ltd, Bangalore, India). The purity of RNA was checked by formaldehyde agarose gel electrophoresis.
RT and PCR were performed in two tubes. First strand cDNA synthesis was performed using Omniscript RT kit (Qiagen Inc., Chatsworth, CA, USA) according to the manufacturer's protocol. Resulting cDNA was stored at -20°C.
PCR product was amplified from the cDNA using primers specific to potyviruses re synthesized from 1st BASE Company (Agile Lifescience Technologies India Pvt. Ltd. Airoli, Navi Mumbai) in salt free status. Forward primer HRP4 5'TGG TGY ATH GAN AAT GG* 3' (*Y: C/T, H: A/T/C, N: A/G/C/T) and reverse primer HRP1 (5' TTT  TTT TTT TTT TTT TTT TTT A 3'), HRP2 (5' TTT TTT TTT TTT  TTT TTT TTT G 3'), HRP3 (5' TTT TTT TTT TTT TTT TTT TTT  C 3'). 25 ml of reaction mixture containing 2.5 μl of cDNA, 20 pmol each of forward and reverse primers, 200 mM each of dNTPs, 2.5 μl of 10X PCR buffer with 15 mM MgCl 2 and 0.5 unit of Taq DNA polymerase (Bangalore Genei Pvt. Ltd, Bangalore, India) were used in PCR. The amplification was performed in a CR Palm cycler (Corbett Research, Australia). Temperature profile used in the amplification was as preincubation at 94°C for 5 min leading to 40 cycles of denaturation at 94°C for 1 min, annealing at 42°C for 2 min and synthesis at 72°C for 90 sec. After amplification, the PCR products were electrophoreses in 1.5% agarose gel.

Symptomatology
Mechanically inoculated seedlings of snake gourd expressed symptoms after 25 days of inoculation. Mosaic, leaf blistering and combinations of these symptoms were observed on leaves of snake gourd seedlings (Figure 1).

Host range
In host range studies, the virus isolate produced chlorotic local lesions on leaves of C. amaranticolor. The symptoms were observed within 15-17 days of inoculation. The virus isolates caused symptoms on all the tested cucurbitaceous hosts as mosaic and chlorosis in Luffa acutangula, mosaic and leaf blisters in Trichosanthes cucumerina and Lagenaria siceraria, mosaic and leaf distortion in Cucumis sativus,   Figure 2).

Physical properties
Studies on physical properties of the virus isolates revealed that DEP was 10-2 for the isolate from snake gourd respectively. The severity grade at DEP for the virus was 2.0. TIP the virus isolate was found to be 55°C with a severity grade of 1.0. The LIV was 30 h at room temperature (28 ± 2°C) with a severity grade of 2.0 in both the cases.

Electron microscopy
Flexuous rod shaped virus particles measuring 750×12 nm were observed in artificially inoculated leaf samples of snake gourd.

Serology
Results of DAS-ELISA revealed that the isolates showed a positive reaction with PRSV antiserum. The OD value for infected samples of snake gourd was recorded 0.566 with PRSV antiserum at 405 nm. For PRSV positive samples it was 0.865. The absorbance values for buffer, healthy samples were 0.116 and 0.204 respectively. There was no reaction with CMV and TSV antisera as the absorbance for healthy and infected samples were in similar range.

Molecular characterization of virus isolates
Total RNA extracted from infected and healthy leaves of snake gourd exhibited two prominent bands of 5 Kb (28s rRNA) and 2 Kb (18s rRNA) and one light band of small RNA. In RT-PCR with the potyvirus specific primers HRP 1, HRP 2, HRP 3 and HRP 4 an amplicon of ~650 bp was obtained. There was no amplification in healthy samples (Figure 3).

Discussion
As the snake gourd is an important cucurbit grown in the area Tamil Nadu and surrounding. This crop cultivation threatens by mosaic disease which produces mosaic, mosaic mottling, leaf blistering and combinations of these symptoms. To identify and confirm the virus type above study has been conducted as a post-graduation research.
Since the isolates of the present investigation failed to produce symptoms on fabaceous, solanaceous hosts and H. annuus, it was concluded that the virus under study was not CMV. The isolates of our study caused systemic symptoms on cucurbits as mosaic mottling and blistering on bottle gourd and snake gourd; mosaic, leaf distortion and leaf blisters on cucumber and melon etc. This is in corroboration with the findings of Roy et al. [8] who observed similar symptoms for the   PRSV-W isolate from pumpkin. However, no symptom was observed in his studies on C. amaranticolor while chlorotic local lesions were induced by our isolates in the present research.
Further with the serology analysis isolates of snake gourd from our study showed positive reactions with PRSV antiserum, while no reaction was observed with CMV and Tobacco streak virus antisera. The PRSV-W isolate of pumpkin and zucchini was found to have close reaction with PRSV antiserum and did not react with ZYMV, CMV and WMV antisera [3].
Finally the RT-PCR of RNA from infected snake gourd samples with potyvirus specific reverse primers HRP1, HRP2, HPR3 and forward primer HRP4 yielded an amplicon of ~650 bp which represents the posterior half CP gene of PRSV. Jain et al. [9] used the same reverse primers with HRP 10, HRP 51 and HRP 52 as forward primers in several combinations to amplify a part of NIb gene, complete CP gene and UTR of PRSV. A product of 1.7 Kb was obtained and sequenced. An amplicon of >1Kb representing the CP and part of NIb gene of PRSV-P was obtained by Sharma et al. [10] with primer HRP 50 and RKJ 3. Sharma et al. [11] obtained a ~850 bp product from papaya infected with PRSV-P using primers HRP 52 and RKJ 3 that were specific for amplifying the CP gene. Jain et al. [9] in their attempt to clone the CP gene of PRSV-W had observed a 655 bp sequence region which includes the primer sequence of HRP 4.
Based on biological, physical and morphological properties, serology and molecular characterization of the virus isolates which caused mosaic syndrome in snake gourd were concluded as isolates of PRSV-W.