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Pcr-based Detection and Characterization of Papaya Viruses in Bangladesh (2019--2020)

M.A.Y. Akhond.
Biotechnology Division, BARI, Joydebpur, Gazipu

K. Nahar Biotechnology Division, BARI, Joydebpur, Gazipu

N. Bilkish
Biotechnology Division, BARI, Joydebpur, Gazipu

S. Ahmed
Biotechnology Division, BARI, Joydebpur, Gazipu

Abstract/ Executive Summary:

Papaya (Carica papaya, Caricaceae) is a major horticultural crop in Bangladesh. The tree is often infected by various pests and diseases. Papaya ringspot virus (PRSV) is the cause of one of the most important diseases of the plant which is transmitted by aphids. A total of 34 papaya leaf samples from 12 districts of Bangladesh were collected from papaya plants showing various types of symptoms consistent with virus infection. Total RNA was extracted from all of those and 32 samples were analyzed by Reverse Transcription Polymerase Chain Reaction (RT-PCR) method. Twenty-seven samples were found to be RT-PCR positive for Papaya ringspot virus (PRSV). Complete coat protein (CP) gene sequence was obtained from 16. Phylogenetic analysis of the 16 isolates based on the CP gene showed two major clusters having high polymorphism among the virus isolates within the clusters.

Keywords: Transgenic papaya, Molecular techniques, PCR-based detection

Location:

Start Date: 00-00-2019

End Date: 00-00-2020

Program Area: Variety and Species

Commodity: Papaya

Objectives:

The present research was undertaken to identify the papaya infecting virus/es in Bangladesh using advanced molecular techniques. The information obtained from the research could be used in the future to develop virus-resistant transgenic papaya.

Methodology:

Virus-infected papaya leaf samples were collected from 12 districts of Bangladesh. DNA and RNA were extracted from all the collected samples and preserved at -80°C for further analyses. Various virus-specific primers were designed in-house or collected from published literature and synthesized by commercial service providers (Bio Basic, Canada). Direct PCR amplification was performed using the isolated DNA samples as a template and one or two-step RT-PCR was performed using the isolated RNA samples as a template. The amplified products were electrophoresed in 1% agarose gel. Products showing bands of expected sizes were purified and sequenced on both strands. Identities of the sequenced isolates were confirmed by using BLAST. All the sequences were aligned using the ‘Muscle’ software and a phylogenetic tree was constructed for comparison. Sequence alignment and the tree construction using the Maximum likelihood method were carried out from within the sequence analysis software (MEGA7) based on the Tamura-Nei model (1993). The evolutionary history was inferred by using the Maximum Likelihood method based on the Tamura-Nei model [Tamura K. and Nei M. (1993).]. The bootstrap consensus tree inferred from 100 replicates (Felsenstein J., 1985) is taken to represent the evolutionary history of the taxa analyzed (Felsenstein J., 1985). Branches corresponding to partitions reproduced in less than 50% bootstrap replicates are collapsed. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (100 replicates) are shown next to the branches (Felsenstein J., 1985). Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach and then selecting the topology with a superior log-likelihood value. The analysis involved 16 nucleotide sequences. Codon positions included were 1st+2nd+3rd+Noncoding. All positions containing gaps and missing data were eliminated. There were a total of 504 positions in the final dataset. Evolutionary analyses were conducted in MEGA7 (Kumar et.al. 2016).

Source of Information: Annual Research Report 2019-2020, Biotechnology Division, BARI, Joydebpur, Gazipur

Article Link (Online Journal):

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

A total of 34 papaya leaf samples from 12 districts of Bangladesh were collected from papaya plants showing various types of symptoms consistent with virus infection. Total RNA was extracted from all of those and 32 samples were analyzed by Reverse Transcription Polymerase Chain Reaction (RT-PCR) method. Amplification products were obtained from RT-PCR using primers specific for the Papaya ringspot virus (PRSV). Primers specific for several other viruses failed to produce any amplification products either from PCR or from RT-PCR. Twenty-seven samples were found to be RT-PCR positive for Papaya ringspot virus (PRSV). Complete coat protein (CP) gene sequence was obtained from 16 isolates and sequencing of the CP gene of 11 samples is yet to be completed. Phylogenetic analysis of the 16 isolates based on the CP gene showed two major clusters having high polymorphism among the virus isolates within the clusters. The result is in agreement with that reported previously by Hamim et al. 2019. However, further study with a larger dataset is required for understanding the full extent of genetic diversity in PRSV in Bangladesh. The experiment will be continued for characterizing the rest of the collected isolates or for including more isolates if required.

Knowledge Management: Report/Proceedings