MOLECULAR CHARACTERIZATION AND SERODIAGNOSIS OF PHYTOPHTHORA AND TRISTEZA VIRUS IN CITRUS
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Date
2017-06-15
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Dr. Panjabrao Deshmukh Krishi Vidyapeeth, Akola
Abstract
The present investigation entitled “Molecular characterization and serodiagnosis of Phytophthora and tristeza virus in citrus” was carried out at Molecular breeding Laboratory, Biotechnology Center, Department of Agricultural Botany, Post Graduate Institute, Dr. Panjabrao Deshmukh Krishi Vidyapeeth, Akola.
Citrus is one of the major horticultural crops of India. In recent years, the production of citrus group is greatly affected by a disease like Phytophthora and CTV. This pathogen causes economic damage to the field. For management of a disease there is need to have diagnosis method which should be accurate, rapid, easy and cost effective. So the present study was designed to develop a molecular and immunological diagnosis method for Phytophthora and CTV at field as well as lab level.
The Phytophthora was isolated from soil samples of symptomatic orchards of vidarbha region by using leaf bait method and specific media of CMA-PARPH. The isolates of Phytophthora were confirmed by morphologically. It was found that P. nicotianae was prominently present followed by P. palmivora in Vidarbha region of Maharashtra.
Polyclonal antibodies were produced against Phytophthora and CTV in rabbit using purified Phytophthora mycelial growth and ultrapurified virus respectively. The raised antiserum was confirmed using Dot-ELISA. Polyclonal antibodies detected closely related species during assay. For standardization of immunological diagnosis methods on the basis of antiserum, monoclonal antibodies were used for accuracy.
In Direct Dot-ELISA at the titer dilution were recorded as 1:20 of antigen 1:16000 of primary antibody and 1:2000 of secondary antibody. In indirect Dot-ELISA the titer dilution were recorded as 1:10 of antigen 1:8000 of primary antibody and 1:2000 of secondary antibody. No colour was seen in control showing the sensitivity detection of the assay. In Direct Plate ELISA for Phytophthora the titer was fixed at 1:20 of antigen, 1:16000 of primary antibody and 1:1000 of secondary antibody. The cut off value was confirmed as 0.711 at 1:1000 of secondary antibody and 0.868 at 1:2000 of secondary antibody dilution. In indirect Plate ELISA for Phytophthora the titer was fixed at 1:50 of antigen, 1:16000 of primary antibody and 1:1000 of secondary antibody. No color was observed in control. All values above this cut-off were regarded as potential positive.
In Standardization of incubation period for Dot-ELISA procedure that the optimum colour development was observed at 15 min incubation time.
In molecular detection the DNA was isolated from pure culture of Phytophthora and amplified with series of ITS primer for amplification. From all ITS the ITS4-ITS6, ITS1-ITS4, ITS1-ITS2, ITS4-ITS5 combinations were used to carry out experiment for detection of Phytophthora from pure culture as well as infected soil sample. Discrimination assay was carried out using ITS across the other fungi i.e. Fusarium spp., Rhizoctonia spp., Trichoderma spp., Aspergillus spp., Alternaria spp., Sclerotium spp. which are majorly found fungal species in citrus orchard. Restriction digestion profiling was carried out for detection and discrimination of Phytophthora from other fungi. The restriction enzymes examined BamH-I, Bsu-I, Hpa-I, Sma-I, Hinf-I, Hpa-II, Mva-I Pst-ITaq-I, Dra-I, Pvu-I, Hha-I, Alu-I, Hind-III, Msp-I, EcrR-I, Bsp-I, Hpa-I, Hha-I and Pvu-I were used for the species discrimination in different locations and with different species.
In sensitivity assay the ITS4-ITS5 detects the intensity of DNA from sample up to 100 ag and about 1 ag by D-E set of primer. It concludes that sequence based marker were more sensitive than others.
The CTV infected samples were collected from citrus plants showing characteristic symptoms of CTV as vein clearing, quick decline, Seedling yellowing, Stunting, Vein corking, Epinasty, Leaf cupping was observed in vidarbha region. The virus was isolated by ultracentrifugation with cesium sulphate and sucrose gradient. The electron microscopic studies of CTV revealed the flexuous rod shaped particles measuring 2000 nm in length and 12 nm in diameter.
The indexed seedlings produced characteristic disease symptoms of seedling yellowing, epinasty and leaf cupping after two to four month inoculation of CTV and subsequently growth was reduced, leaving the plants with a dwarfed appearance, shorter stem, internodes, petioles and underdeveloped. This was carried out for biological testing of CTV using indicator plants.
In Direct Dot- ELISA the titer dilution were recorded at 1:20 of antigen 1:16000 of primary antibody and 1:2000 of secondary antibody. No colour was seen in control showing the sensitivity detection of the assay. For detection of CTV on field this method was preferred.
In Direct Plate ELISA titer was fixed at 1:10 antigen and 1:16000 dilutions of antisera with secondary antibody dilution of 1:1000. The cut off value was confirmed as 0.188 at 1:1000 of secondary antibody and 0.189 at 1:2000 of secondary antibody dilution. In Indirect Plate ELISA the titer was fixed 1:10 of antigen, 1:16000 of primary antibody and 1:1000 of secondary antibody. No color was observed in control. The cut off value was confirmed as 0.157 at 1:1000 of secondary antibody and 0.124 at 1:2000 of secondary antibody dilution. All values above this cut-off were regarded as potential positive for CTV.
For molecular detection of CTV, viral RNA was isolated from the symptomatic leaf tissue using Trizol reagent method. Coat Protein gene specific primers were used for amplification of CP gene and ~ 672 bp amplicon size of CP gene of CTV. The CP genes isolated from different host were sequenced for molecular characterization and detection of virus. The primers were designed for molecular detection as well as standardization of Real time-PCR assay using sequenced data. So the set of primers i.e. BTC-CTV series were designed and validated. From the available nucleotide sequence the amino acid of coat protein was designed using Ex-pasy tools and 3D structure of Coat protein was designed using SWISS Model and validated by Ramchandran plot.
So the standardized protocol as well as designed primers and cutoff values will be used further in the detection of Phytophthora and CTV diseases in citrus.
Description
In the present study entitled molecular characterization and serodiagnosis of Phytophthora and tristeza virus in citrus the researcher has tried to find out the diagnosis method for Phytophthora and CTV; major diseases of citrus and help in the accurate management.
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Citation
DEEPA NAVANATH MUSKE. (2017). Molecular characterization and serodiagnosis of Phytophthora and tristeza virus in citrus. Department of Agricultural Botany. Dr. Panjabrao Deshmukh Krishi Vidyapeeth, Ph. D. 2017, 223p, (Unpublished).