Investigation of ORF0 as a sensitive alternative diagnostic segment to detect Sugarcane yellow leaf virus (original) (raw)
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Australasian Plant Pathology, 2017
Yellow leaf (YL) of sugarcane caused by Sugarcane yellow leaf virus (SCYLV) is a serious disease affecting production and productivity in many ruling sugarcane varieties in India, especially in coastal region of Andhra Pradesh state, causing losses ranging from 60% in the first crop to 100% in the ratoon crop. During 2013-2015 seasons SCYLV indexation of meristem tip culture derived sugarcane plantlets as well as usual seed canes planted field varieties was carried out using DAS-ELISA and RT-PCR at RARS, Anakapalle, India. Both leaf extracts and stem sap samples were used. Sixteen samples out of 20 were positive to DAS-ELISA SCYLV diagnosis with OD 405 values of 0.707 to 1.788, while only 13 samples had the visible disease symptoms out of the 20 samples comprising 13 different varieties. This proved efficacy of DAS-ELISAfor indexing even asymptomatic YL infected sugarcane plants. RT-PCR test also detected the virus in both symptomatic and asymptomatic plants. The detection efficiency was considerably good with samples obtained from the same plant (both stem sap and leaf extract) either with colour change or with OD 405 values proving effective and quick indexing using DAS-ELISA. Further, RT-PCR can be used as a confirmatory test to determine the sensitivity of ELISA. In case of in vitro regenerated sugarcane plantlets, all the samples, both hardening stage plantlets and field grown meristem tip culture derived plants didn't showed any positive reaction for the virus daignosed by DAS-ELISA with lower OD 405 values of 0.082 to 0.0321compared to disease free field varieties (OD 405 from 0.151 to 0.355) making it as best management option with proper indexing.
Australasian Plant Pathology
Thirty sugarcane leaf samples exhibiting midrib yellowing symptoms from nine sugarcane growing states of India were collected. The total RNA was isolated from infected samples and RT-PCR assays were performed using Sugarcane yellow leaf virus (SCYLV) specific primers. The infection of SCYLV was detected in 27 out of 30 samples, which showed the expected size (~610 bp) amplicon during RT-PCR. The amplicons from 13 samples were cloned, sequenced and sequence data of these isolates were analyzed to determine sequence identities, sequence variations and phylogenetic relationships with the SCYLV isolates reported earlier from India and abroad with the view to identify the virus isolates based on partial sequence of ORF5, and complete ORF3 (coat protein, CP) and ORF4 (movement protein, MP). The analysis of the complete sequence data (613 nucleotides) of 13 isolates under study revealed 96–100% identities among them and also with SCYLV-Cuban isolate. The identities were 90–94% with other isolates of SCYLV reported from other countries. The nucleotide (nt) and amino acid (aa) sequence comparison in the CP and MP coding regions showed a significant variation between Indian isolates (SCYLV-IND) and other SCYLV isolates reported worldwide. Phylogenetic analyses of the 13 SCYLV isolates under study showed that they clustered together along with most other SCYLV-Indian isolates and a SCYLV-Cuba isolate. The remaining isolates from other regions of the world and a single isolate from India characterized in a previous study clustered into separate groups. Therefore, the isolates under study were identified as isolates of SCYLV which are closely related to SCYLV-Cuba, the member of ‘CUB’ genotype.
European journal of plant …, 2010
Sugarcane yellow leaf virus (SCYLV) has been reported worldwide to infect sugarcane and to cause significant yield losses. Current detection methods include tissue blot immunoassay (TBIA), reverse transcription-polymerase chain reaction (RT-PCR) and quantitative real-time RT-PCR assay (qRT-PCR). In this paper, we report the use and comparison of these detection methods for the study of SCYLV in Hawaiian cultivars. We observed positive RT-PCR and qRT-PCR reactions in cultivars previously thought (based on TBIA) to be immune to virus infection. The semi-quantitative virus titre in these cultivars was however at least 10 6-fold lower than in the cultivars which were known to be SCYLVsusceptible. The RT-PCR methods also revealed that plants of the cultivar H65-7052, which were previously shown to vary strongly between TBIA-positive and TBIA-negative, indeed exhibited fluctuating SCYLV-titres in a range of 10 3-10 4-fold. The virus titre was carried through to the next vegetative generation, i. e. plants grown from seed pieces with low virus titre had low virus titre and plants grown from seed pieces with high virus titre contained high virus titre. A small field trial comparing plants of cv. H65-7052 of low and high SCYLV-titre showed that the field plots with plants of high virus titre developed Yellow Leaf symptoms and yielded only 54-60% of cane and sugar tonnage compared to plots with plants of low virus titre.
Molecular evidence that sugarcane yellow leaf virus (ScYLV) is amember of the Luteoviridae family
Archives of Virology, 2000
A previously uncharacterized virus was reported in southeast Brazil causing a yellowing leaf disease in sugarcane. The virus, termed sugarcane yellow leaf virus (ScYLV), shares features typical of the luteoviruses. To start the molecular characterization of ScYLV, the nucleotide sequence of the coat protein (CP), 17 kDa protein and C-terminus of the RNA-dependent RNA polymerase coding regions was determined from an RT-PCR amplification product. Comparisons showed that the deduced amino acid sequences share a considerable degree of identity and similarity with corresponding sequences of known luteoviruses, thus clearly establishing ScYLV as a member of the family Luteoviridae. The authenticity of the CP open reading frame was confirmed by its expression in Escherichia coli. The recombinant CP positively reacted in immunoblot assays with polyclonal antibodies raised against native ScYLV. Furthermore, phylogenetic analyses also suggest that the 5′ and 3′ coding blocks of the ScYLV genome possess different taxonomic affinities within the Luteoviridae family, as does also the genome of soybean dwarf virus.
Diagnosis of Sugarcane yellow leaf virus in asymptomatic sugarcane by RT-PCR
Sugar Tech, 2009
Sugarcane yellow leaf virus (SCYLV) that causes yellow leaf disease (YLD) in sugarcane belongs to Polerovirus. The disease expression is commonly observed during 6 to 8 months stages of the crop and there is a need to diagnose the disease before the symptom appearance. We have standardized RT-PCR technique with a new set of primers to detect SCYLV in sugarcane in pre-symptomatic stages of YLD. During presymptomatic stage 33 of the 44 varieties studied tested positive to SCYLV in RT-PCR. During eighth month stage all the 44 varieties have shown characteristic disease symptoms and except one all of them tested positive to the virus in RT-PCR. The two RT-PCR assays performed separately during pre-and post-symptom expression stages in 44 varieties clearly revealed that most of the varieties have detectable level of the virus in asymptomatic stage. Expression of disease symptoms and presence of the virus in all the varieties except one very clearly indicated the severe virus infection in the tested varieties. The studies also proved the diagnostic efficiency of the new set of primers to detect SCYLV in asymptomatic plants.
The Plant Pathology Journal
Sugarcane yellow leaf virus (SCYLV) is a distinct member of the Polerovirus genus of the Luteoviridae family. SCYLV is the major limitation to sugarcane production worldwide and presently occurring in most of the sugarcane growing countries. SCYLV having high genetic diversity within the species and presently ten genotypes are known to occur based on the complete genome sequence information. SCYLV is present in almost all the states of India where sugarcane is grown. Virion comprises of 180 coat protein units and are 24-29 nm in diameter. The genome of SCYLV is a monopartite and comprised of single-stranded (ss) positive-sense (+) linear RNA of about 6 kb in size. Virus genome consists of six open reading frames (ORFs) that are expressed by sub-genomic RNAs. The SCYLV is phloem-limited and transmitted by sugarcane aphid Melanaphis sacchari in a circulative and non-propagative manner. The other aphid species namely, Ceratovacuna lanigera, Rhopalosiphum rufiabdominalis, and R. maidis also been reported to transmit the virus. The virus is not transmitted mechanically, therefore, its transmission by M. sacchari has been studied in different countries. SCYLV has a limited natural host range and mainly infect sugarcane (Sachharum hybrid), grain sorghum (Sorghum bicolor), and Columbus grass (Sorghum almum). Recent insights in the protein-protein interactions of Polerovirus through protein interaction reporter (PIR) technology enable us to understand viral encoded proteins during virus replication, assembly, plant defence mechanism, short and long-distance travel of the virus. This review presents the recent understandings on virus biology, diagnosis, genetic diversity, virus-vector and host-virus interactions and conventional and next generation management approaches.
European Journal of Plant Pathology, 2010
Sugarcane yellow leaf virus (SCYLV) has been reported worldwide to infect sugarcane and to cause significant yield losses. Current detection methods include tissue blot immunoassay (TBIA), reverse transcription-polymerase chain reaction (RT-PCR) and quantitative real-time RT-PCR assay (qRT-PCR). In this paper, we report the use and comparison of these detection methods for the study of SCYLV in Hawaiian cultivars. We observed positive RT-PCR and qRT-PCR reactions in cultivars previously thought (based on TBIA) to be immune to virus infection. The semi-quantitative virus titre in these cultivars was however at least 10 6-fold lower than in the cultivars which were known to be SCYLVsusceptible. The RT-PCR methods also revealed that plants of the cultivar H65-7052, which were previously shown to vary strongly between TBIA-positive and TBIA-negative, indeed exhibited fluctuating SCYLV-titres in a range of 10 3-10 4-fold. The virus titre was carried through to the next vegetative generation, i. e. plants grown from seed pieces with low virus titre had low virus titre and plants grown from seed pieces with high virus titre contained high virus titre. A small field trial comparing plants of cv. H65-7052 of low and high SCYLV-titre showed that the field plots with plants of high virus titre developed Yellow Leaf symptoms and yielded only 54-60% of cane and sugar tonnage compared to plots with plants of low virus titre.