Development of RT-PCR degenerate primers for the detection of two mandariviruses infecting citrus cultivars in India.

Indian citrus ringspot virus (ICRSV) and Citrus yellow vein clearing virus (CYVCV) are the mandariviruses infecting various citrus cultivars in India and around the world. In the fields, it was observed that citrus plants infected by both the viruses and frequently expressed only ringspot symptoms. The ICRSV-specific polyclonal-antibody used in immuno-sorbent electron microscopy (ISEM) and enzyme linked immuno-sorbent assay (ELISA) could detect only ICRSV in mixed infections. Therefore, the conserved sequences of the RNA dependent RNA polymerase (RdRP) gene of the alphaflexiviruses were exploited for developing a RT-PCR based assay for detection of both the mandariviruses simultaneously, if present. A degenerate primer pair was designed to amplify a ∼435bp fragment by multiple alignments of the RdRP gene sequences of the members of genera Mandarivirus, Potexvirus and Allexivirus. The developed RT-PCR assay was validated for detecting both, CYVCV and ICRSV in mixed infections as well as in single virus-infected citrus plants. The presence of ICRSV or CYVCV or both of them together in such plants were confirmed by using primer pair specific to each of these viruses. Further, the identity of the amplicons was confirmed by sequencing and the virus species were determined with BLASTN analysis. The degenerate primers also amplified the corresponding target sequences of an allexivirus and a potexvirus from the respective infected garlic/ onion and tobacco plants. The use of the degenerate primers for the detection of these virus species of the genus Mandarivirus will be useful in citrus certification programmes.

Genome characterization of citrus yellow vein-clearing virus: limited heterogeneity of viral genomes in Mandarivirus-infecting different citrus species.

Citrus yellow vein-clearing virus (CYVCV) is a mandarivirus infecting citrus producing yellow vein-clearing symptoms. The leaf samples collected during surveys of different citrus-growing areas in India exhibited diverse symptoms and 40% of the plants were positive for CYVCV in RT-PCR, indicating the wide distribution of the virus in India. It was reported for first time that CYVCV infects kinnow mandarin and sweet oranges and produces chlorotic ringspots symptoms identical to Indian citrus ringspot virus (ICRSV). The complete genome sequences of CYVCV infecting four citrus cultivars have been deciphered through overlapping primers. All the four genomes comprise of 7531 nucleotides excluding the 3' poly (A) tail. The sequence identity of genomes of four CYVCV isolates in the present study ranged from 95.2 to 99.8% with genome sequences of 31 CYVCV isolates available in public domain and the mean genomic diversity was 0.017, indicating low level of heterogeneity. The phylogenetic analysis revealed that CYVCV isolates from India, Pakistan, and Turkey were clustered in the same clad apart from China isolates. The least normalized dN/dS mean value (0.092) indicated that RdRP region evolved under relatively stronger selection constraints than the other five coding regions of CYVCV. The four intragenic putative recombination events detected in RDP4 program occurred naturally in CYVCV genome, indicating the evolutionary progress of the virus. Tajima's and Fu and Li's D parameters were performed using genomic sequences in DnaSP v5 program and the retrieved negative values indicated the presence of limited genetic variability in CYVCV genomes. To the best of our knowledge, this is the first comprehensive report on molecular characterization of CYVCV from India. It will be helpful in understanding the evolutionary relationship of CYVCV and ICRSV.

First report of cucumber mosaic virus infecting antamul vine (Tylophora indica) in India.

Tylophora indica (Burm f.) Merrill; commonly known as antamul, is an important medicinal herb. Typical yellow rings or irregular yellow spot and in severe condition necrotic rings were observed on the leaves of the crop. The examination of symptomatic leaf samples under transmission electron microscopy revealed the presence of spherical virions confirmed the association of cucumo like virus group. A novel degenerate primers pair was designed by multiple sequence alignment of RdRP region and used in RT-PCR to amplify a ~ 410 bp genomic fragment. The sequence of the amplified fragment shared 97-98% sequence identity with cucumber mosaic virus (CMV). This study is the first report of the association of CMV with the yellow ring symptom of antamul in India.

Development of multiplex polymerase chain reaction assay for simultaneous detection of clostero-, badna- and mandari-viruses along with huanglongbing bacterium in citrus trees.

Citrus trees harbor a large number of viral and bacterial pathogens. Citrus yellow vein clearing virus (CYVCV), Indian citrus ringspot virus (ICRSV), Citrus yellow mosaic virus (CYMV), Citrus tristeza virus (CTV) and a bacterium, Candidatus Liberibacter asiaticus (CLa) associated with huanglongbing (HLB) disease, the most prevalent pathogens in citrus orchards of different regions in India and are responsible for debilitating citriculture. For detection of these viral and bacterial pathogens a quick, sensitive and cost effective detection method is required. With this objective a multiplex polymerase chain reaction (mPCR) assay was developed for simultaneous detection of four viruses and a bacterium in citrus. Several sets of primers were designed for each virus based on the retrieved reference sequences from the GenBank. A primer pair published previously was used for greening bacterium. Each pair of primers was evaluated for their sensitivity and differentiation by simplex and mPCR. The constant amplified products were identified on the basis of molecular size in mPCR and were compared with standard PCR. The amplicons were cloned and results were confirmed with sequencing analysis. The mPCR assay was validated using naturally infected field samples for one or more citrus viruses and the huanglongbing bacterium. The mPCR assay developed here will aid in the production of virus free planting materials and rapid indexing for certification of citrus budwood programme.