Sugarcane Streak Mosaic Virus Research Articles

Expression of Recombinant Sugarcane Streak Mosaic Virus Coat Protein Gene in Escherichia coli

Sugarcane streak mosaic virus (SCSMV) is an important virus causing mosaic disease in sugarcane and transmitted through the cutting cane. Commercial antiserum to detect SCSMV and to monitor the disease development is not available. The research was conducted to produce antigen of SCSMV coat protein (SCSMV-CP) through overexpressing it on bacterial expression which will be used for antiserum production. SCSMV-CP was amplified using specific primers for CP gene containing BamHI and HindIII restriction enzyme sites and cloned into pTZ57R/T. Subsequently, the SCSMV-CP was subcloned into pET28a and transformed on Escherichia coli BL21(DE3) and Rosetta-gami(DE3)pLysS. The concentration of isopropyl β-d-thiogalactopyranoside (IPTG), incubation temperature, and bacterial harvesting time after IPTG induction were optimized. SCSMV-CP gene was successfully amplified with size ±855 bp, subcloned into vector expression, and expressed in insoluble fraction either in both bacterial host. Optimal protein expression of SCSMV-CP recombinant was obtained at 25°C with IPTG concentration 0.25–1.00 mM and harvested at 9–12 hours after IPTG induction in E. coli BL21(DE3), and at 30°C with IPTG concentration 0.25–1.00 mM and harvested 3–12 hours after IPTG induction in E. coli Rosetta(DE3)pLysS. SDS-PAGE analysis showed that protein size of SCSMV-CP recombinant was ±35.4 kDa.

Cis-proteolytic activity of a recombinant nuclear inclusion a (NIa) proteinase from Sugarcane Streak Mosaic Virus, a member of the genus Poacevirus in the family Potyviridae

The complete genome of Sugarcane streak mosaic virus-Andhra Pradesh isolate (SCSMV-AP), a member belonging to the genus Poacevirus of the family Potyviridae was previously sequenced. Among the non-structural proteins, nuclear inclusion protein a proteinase (NIa-Pro) is a multifunctional protein that plays an important role in the life cycle of Potyviridae members. In this study, N-terminal hexahistidine (His6)-tagged NIa-Pro gene of SCSMV-AP was amplified by RT-PCR, cloned, and expressed in Escherichia coli. Recombinant NIa-Pro was purified by Ni-NTA affinity chromatography and used to raise polyclonal antibodies. cis-Proteolytic activity of NIa-Pro was confirmed in vitro using a recombinant polyprotein substrate containing the virus protein genome-linked (VPg) and NIa cleavage site. Mutant VPgNIa constructs were generated by site-directed mutagenesis to investigate the role of conserved amino acid residues H242, D277 and H204 in the catalytic process of NIa proteinase. Such an analysis revealed that H242 and D277 constitute two of the predicted amino acid residues of the catalytic traid and H204 is probably a crucial amino acid at P1 position and confirmed the predicted unusual cleavage site (H/A) between VPg and NIa of SCSMV polyprotein.

Sugarcane streak mosaic virus. [Distribution map

Abstract A new distribution map is provided for Sugarcane streak mosaic virus. Potyviridae: Poacevirus. Main host: sugarcane ( Saccharum officinarum ). Information is given on the geographical distribution in Asia (Bangladesh, China, Guangdong, Guangxi, Hainan, Yunnan, India, Andhra Pradesh, Assam, Bihar, Haryana, Karnataka, Kerala, Madhya Pradesh, Maharashtra, Indian Punjab, Tamil Nadu, Uttar Pradesh, Uttarakhand, Indonesia, Java, Iran, Japan, Pakistan, Sri Lanka, Thailand and Vietnam).

Genomic variability and molecular evolution of Asian isolates of sugarcane streak mosaic virus.

Sugarcane streak mosaic virus (SCSMV), an economically important causal agent of mosaic disease of sugarcane, is a member of the newly created genus Poacevirus in the family Potyviridae. In this study, we report the molecular characterization of three new SCSMV isolates from China (YN-YZ211 and HN-YZ49) and Myanmar (MYA-Formosa) and their genetic variation and phylogenetic relationship to SCSMV isolates from Asia and the type members of the family Potyviridae. The complete genome of each of the three isolates was determined to be 9781 nucleotides (nt) in size, excluding the 3' poly(A) tail. Phylogenetic analysis of the complete polyprotein amino acid (aa) sequences (3130 aa) revealed that all SCSMV isolates clustered into a phylogroup specific to the genus Poacevirus and formed two distinct clades designated as group I and group II. Isolates YN-YZ211, HN-YZ49 and MYA-Formosa clustered into group I, sharing 96.8-99.5% and 98.9-99.6% nt (at the complete genomic level) and aa (at the polyprotein level) identity, respectively, among themselves and 81.2-98.8% and 94.0-99.6% nt (at the complete genomic level) and aa (at the polyprotein level) identity, respectively, with the corresponding sequences of seven Asian SCSMV isolates. Population genetic analysis revealed greater between-group (0.190±0.004) than within-group (group I=0.025±0.001 and group II=0.071±0.003) evolutionary divergence values, further supporting the results of the phylogenetic analysis. Further analysis indicated that natural selection might have contributed to the evolution of isolates belonging to the two identified SCSMV clades, with infrequent genetic exchanges occurring between them over time. These findings provide a comprehensive analysis of the population genetic structure and driving forces for the evolution of SCSMV with implications for global exchange of sugarcane germplasm.

Characterization and Genetic Variation of Sugarcane Streak Mosaic Virus, a Poacevirus Infecting Sugarcane in Thailand

<p>Sugarcane disease surveys were conducted from 2010 to 2014 at major sugarcane growing areas in 5 provinces (Nakhon Pathom, Kanchanaburi, Udon Thani, Khon Kaen, Nakhon Ratchasima) and germplasm collection fields. Random samples of the virus-like sugarcane leaves obtained from the surveyed areas suggested yellow streak mosaic symptoms. Direct antigen coating ELISA using locally produced SCSMV antiserum, revealed widespread incidence of SCSMV in the major sugarcane growing areas and the germplasm collection fields, ranging from 43.48-90.91% and 54.17-100% respectively. The virus isolate from sugarcane in Kamphaeng Saen, Nakhon Pathom, designated as THA-NP3, was characterized by genomic sequencing. Complete genome of THA-NP3 (JN163911) contained 9,781 nucleotides, excluding 3¢ Poly (A) tail which encoded a polyprotein of 3,130 amino acid residues comprising 10 functional proteins, namely P1, HC-Pro, P3, 6K1, CI, 6K2, NIa-VPg, NIa-Pro, NIb and CP. Sequence comparisons revealed that THA-NP3 showed 97.84% nucleotide identity to JP2 (JF488065) from China and 81.39-97.78% nucleotide identities to other recorded SCSMV sequences. Detection for the presence of CP gene by RT-PCR indicated 1094 bp containing 846 bp of the CP coding region. Analysis of the CP gene revealed genetic variation of 58 Thai SCSMV isolates, 86.17-100% nucleotide identities among them and 85.70-99.29% nucleotide identities to SCSMV isolates from other countries. Recombination events existed in the CP coding regions between two distinct sub-populations, the germplasm isolates and the farmers’ field isolates. These results suggested the incidence of SCSMV variants between the farmers’ fields and the germplasm collection fields. <strong></strong></p>

Genetic structure of populations of sugarcane streak mosaic virus in China: Comparison with the populations in India

Sugarcane streak mosaic virus (SCSMV) causes mosaic and streak symptoms on sugarcane and sorghum crops, and has a broad host range. SCSMV is a member of the genus Poacevirus in the family Potyviridae.Ten SCSMV isolates were collected from sugarcane plants showing mosaic and streaking in Southern China from 2009–2011. Sequence-based phylogenetic and population genetic analyses were conducted using four partial genomic sequences covering the full genomes. These analyses were used to estimate the subpopulation differentiation and divergence within the Chinese virus population, and were compared with isolates from India. SCSMV-infected sugarcane plants in the field commonly harbor virus quasispecies (mutant cloud), and often have mixed infections with the same virus isolates. Inter- and intra-lineage recombination sites were identified in the protein 1, helper-component proteinase, coat protein and 3′ non-coding regions of the Chinese isolates. All the Chinese non-recombinant isolates fell into at least nine lineages, and many clustered with Indian isolates. However, estimates of genetic differentiation and gene flow indicated that the SCSMV populations in China and India are genetically independent. Our genetic study of a poacevirus population in South Asia regions indicates the importance of the evolutionary-based design to control viruses.

Sugarcane Elongin C is involved in infection by sugarcane mosaic disease pathogens

Sugarcane (Saccharum sp. hybrid) provides the main source of sugar for humans. Sugarcane mosaic disease (SMD) is a major threat to sugarcane production. Currently, control of SMD is mainly dependent on breeding resistant cultivars through hybridization, which is time-consuming. Understanding the mechanism of viral infection may facilitate novel strategies to breed cultivars resistant to SMD and to control the disease. In this study, a wide interaction was detected between the viral VPg protein and host proteins. Several genes were screened from sugarcane cDNA library that could interact with Sugarcane streak mosaic virus VPg, including SceIF4E1 and ScELC. ScELC was predicted to be a cytoplasmic protein, but subcellular localization analysis showed it was distributed both in cytoplasmic and nuclear, and interactions were also detected between ScELC and VPg of SCMV or SrMV that reveal ScELC was widely used in the SMD pathogen infection process. ScELC and VPgs interacted in the nucleus, and may function to enhance the viral transcription rate. ScELC also interacted with SceIF4E2 both in the cytoplasm and nucleus, but not with SceIF4E1 and SceIF4E3. These results suggest that ScELC may be essential for the function of SceIF4E2, an isomer of eIF4E.

First report of Sugarcane streak mosaic virus in Iran

Sugarcane is one of the most economically important crops in south-western Iran and viral diseases such as those caused by Sugarcane mosaic virus (SCMV) have caused yield losses in some fields. A survey was conducted in Khuzestan province…

Genetic diversity of viruses associated with sugarcane mosaic disease of sugarcane inter-specific hybrids in China

In this study, we investigated 78 sugarcane samples with severe mosaic symptoms collected from four provinces of southern China: Guangxi, Yunnan, Hainan, and Guangdong, which cover nearly 85 % of commercial sugarcane planting zones in China. Using RT-PCR, sequencing and phylogenetic analysis, we identified 72 hybrid sugarcane samples containing causal agents of sugarcane mosaic disease. Among these, 66 virus isolates were identified as Sorghum mosaic virus (SrMV) (84.6 %), four were identified as Sugarcane streak mosaic virus (SCSMV) (5.1 %), and two were identified as Sugarcane mosaic virus (SCMV) (2.6 %). The isolates of SrMV were classified into three subgroups: I, II, and III with a 95 % similarity level. The two largest subgroups, SrMV I containing 36 sugarcane mosaic diseases isolates (46.2 %), and SrMV III containing 20 isolates (25.6 %), were prevalent in Guangdong and Guangxi provinces; however, SrMV II containing five isolates (6.4 %) did not exhibit any close association with geographical distribution. The popular sugarcane cultivar, ROC22, was found to be infected with all the three subgroup types of SrMV. According to our knowledge, this is the first reported detection of the co-infection of SrMV and SCSMV in Saccharum hybrid (YN-bs-9). Recombination analysis indicated recombination between different isolates mostly occurring in Yunnan and Guangxi provinces among the SrMV I group. This study provides insight into the species diversity and geographical distribution of causal agents of sugarcane mosaic disease, and provides the basis for its identification, prevention, and future control efforts.

Molecular Diversity Analysis of Pretty Interesting Potyviridae ORF (PIPO) Coding Region in Indian Isolates of Sugarcane streak mosaic virus

Sugarcane streak mosaic virus (SCSMV) is a distinct member of Poacevirus in Potyviridae family causing mosaic disease in sugarcane. In this study, we computationally predicted potyvirus specific overlapping ORF, PIPO (~139 amino acid) in P3 cistron of known SCSMV genome in +1 reading frame using MLOGD. The nucleotide variations in P3 and the predicted PIPO were experimentally validated using SCSMV isolates from India. Phylogenetic relatedness of identified sequences (P3, PIPO) reflects strong selection and at least three subpopulations within the strains of SCSMV in India. These finding considerably broaden future investigation of the SCSMV pathogenesis in sugarcane.

Dispersal, Yield Losses and Varietal Resistance of Sugarcane streak mosaic virus (SCSMV) in Indonesia

Dispersal, Yield Losses and Varietal Resistance of Sugarcane streak mosaic virus (SCSMV) in Indonesia

Molecular characterization of Indian Sugarcane streak mosaic virus isolates reveals recombination and negative selection in the P1 gene

Sugarcane streak mosaic virus (SCSMV), a member of the genus Poacevirus is an important viral pathogen affecting sugarcane production in India. The P1 gene of ten Indian isolates was sequenced and compared with previously reported SCSMV isolates. Comparative sequence analysis revealed a high level of diversity in the P1 gene (83–98% nucleotide sequence identity; 87–100% amino acid sequence identity), and the Indian SCSMV isolates were found to be the most variable (up to 9% diversity at the amino acid level). Phylogenetic tree analysis showed clustering of 17 SCSMV isolates into two groups: group I included isolates from India (except SCSMV-TPT) and Pakistan, and group II consisted of isolates from Japan, Indonesia, Thailand and SCSMV-TPT. The results obtained from phylogenetic study were further supported by the different in silico analysis viz. SNPs (single nucleotide polymorphism), INDELs (insertion and deletion) and evolutionary distance analysis. A significant proportion of recombination sites were observed at the N terminal region of P1 gene. Analysis of selection pressure indicated that the P1 gene of the Indian SCSMV isolates is under strong negative or purifying selection. It is likely that recombination identified in Indian SCSMV isolates, along with strong purifying selection, enhances the speed of elimination of deleterious mutations in the P1 gene. The evolutionary processes (recombination and selection pressure) together contributed to the observed genetic diversity and population structure of Indian SCSMV isolates.

Molecular variability of sugarcane streak mosaic virus in China based on an analysis of the P1 and CP protein coding regions

Sequences of the protein 1 (P1) and coat protein (CP) coding regions of 22 sugarcane streak mosaic virus (SCSMV) isolates were determined. Phylogenetic analysis showed that SCSMV had at least three major lineages, and the lineages seemed to reflect geographical origin. The sudden expansions of the Chinese and Indian subpopulations were supported by calculations showing deviations from the neutral equilibrium model for the individual lineages with an overall lack of nucleotide diversity. Our study shows that Chinese and Indian SCSMV isolates are part of a distinct population, and the subpopulations probably reflect founder effects.

Sugarcane streak mosaic virus in Indonesia: Distribution, Characterisation, Yield Losses and Management Approaches

Streak mosaic is a new disease of sugarcane in Indonesia caused by Sugarcane streak mosaic virus (SCSMV). An extensive survey conducted during milling season 2008/2009 at 30 sugar factories (SF) across the Java revealed that about 30 % of observed sugarcane fields of 28 SF were affected by the streak mosaic disease. Most commercial cane cultivars were infected by the virus but the cultivar PS 864 was found most susceptible. RT-PCR detection, using SCSMV coat protein specific gene primers SCSMV-cpF and SCSMV AP3, successfully amplified a 500 bp DNA fragment, suggesting the positive identity of the SCSMV with all the tested symptomatic samples. Protein analysis of the virus confirmed that SCSMV has a coat protein of size approximately 40 kDa and flexuous, filamentous particles about 890 nm in length was observed under an electron microscope. The virus was easily transmitted by infected cane cuttings and mechanically by sap inoculation and cutting knife. Host range test on 23 plant species revealed that maize, sorghum and Dactyloctenium aegyptium were alternative hosts of SCSMV. A preliminary yield loss assessment on PS 864 cultivar revealed that the disease incidence at ≥50 % reduced sugar yield by about 20 %. Hot water treatment of cane cuttings was not able to eliminate the virus in cane stalks but only postponed the appearance of the symptom. Response of 16 commercial cane cultivars to artificially inoculation of SCSMV using an abrasive pad rubbing technique showed that only five cultivars were resistant to the disease.

Identification and validation of sugarcane streak mosaic virus-encoded microRNAs and their targets in sugarcane

Plants have developed several defense mechanisms to cope with various pathogens (bacteria, fungi, virus, and phytoplasma). Among these, RNA interference (RNAi)-mediated defense against viral infection was found to be a major innate immune response. As a counter attack strategy against the host defense, viruses produce suppressors of host RNAi pathway. MicroRNAs (miRNAs) are an abundant class of short (~18-22 nucleotide) non-coding single-stranded RNAs involved in RNAi pathway leading to post-transcriptional regulation of gene expression. Sugarcane streak mosaic virus (SCSMV) is a distinct strain of Potyviridae family which has a single-stranded positive-sense RNA genome causing mosaic disease in sugarcane. In this study, we computationally predicted and experimentally validated the miRNA encoded by the SCSMV genome with detection efficiency of 99.9 % in stem-loop RT-qPCR and predicted their potential gene targets in sugarcane. These sugarcane target genes considerably broaden future investigation of the SCSMV-encoded miRNA function during viral pathogenesis and might be applied as a new strategy for controlling mosaic disease in sugarcane.

A Simple Precipitation Approach for Isolation and Enrichment of Sugarcane Streak Mosaic Virus

Sugarcane streak mosaic virus (SCSMV) is a more prevalent sugarcane infecting virus reducing the total sugar production of the world which necessitates rapid screening and detailed studies on SCSMV pathogenicity. A simple reliable, user friendly approach for purification and enrichment of SCSMV from infected sugarcane leaf tissue was developed as alternative to ultracentrifugation approach. The quality and fold enrichment of viral particle was confirmed by RT-PCR and sequence analysis of SCSMV coat protein region. This approach offers 20.03-fold increase in sensitivity for their detection and high viral: host genome ratio of 102 and 103 in infected leaf tissue and purified viral fraction, respectively.

Multiplex RT-PCR for parallel detection of three RNA viruses associated with stripe and mosaic diseases of sorghum in India

Stripe of sorghum caused by Maize stripe virus (MSpV-Sorg) and mosaic of sorghum caused by Sugarcane streak mosaic virus (SStMV-Sorg) and a newly identified rhabdovirus (tentatively named Indian sorghum mosaic virus-ISMV) are the two prevalent and important viral diseases of sorghum in India. In this study, a multiplex-reverse transcription-polymerase chain reaction (M-RT-PCR) was developed for the simultaneous detection and discrimination of these three taxonomically distinct RNA viruses. Three sets of primers were successfully evaluated for their sensitivity and specificity by both uniplex- and M-RT-PCR by optimizing the conditions for simultaneous detection. The designed primers amplified 962, 749 and 497 bp fragments of MSpV, SStMV and ISMV, respectively. The specificities of multiplex primers were confirmed by sequencing the M-RT-PCR products and the generated sequences of the respective virus isolates were deposited in the GenBank. Using the developed M-RT-PCR, MSpV-Sorg up to 1:1000 dilution while SStMV-Sorg and ISMV up to 1:5000 dilutions of total RNA extracts from respective virus infected sorghum leaf samples were clearly detected. Further, the developed technique was successfully evaluated for detection of above viruses in field-collected sorghum samples with stripe and mosaic symptoms

Development of Duplex-Immunocapture (Duplex-IC) RT-PCR for the Detection of Sugarcane streak mosaic virus and Sugarcane mosaic virus in Sugarcane

Mosaic a major disease of sugarcane caused either by Sugarcane streak mosaic virus (SCSMV) or Sugarcane mosaic virus (SCMV) either alone or in combination. Though many serological techniques are available for the detection of viruses, molecular methods are found to be more sensitive in case of low virus titre. For the conventional molecular assays in reverse transcriptase (RT)-PCR, RNA extraction from sugarcane leaves is a cumbersome process. Hence, we optimized immunocapture (IC) using recombinant antiserum (r-antiserum) developed against SCSMV-coat protein (CP) to trap the virus before reverse transcription and optimized a duplex immunocapture reverse transcription (IC-RT) PCR assay. Subsequently we found that the r-antiserum is found to be sensitive to detect both SCMV and SCSMV. Our study established that r-antiserum developed against SCSMV-CP, trapped both SCSMV and SCMV in IC-RT-PCR and it was sensitive enough to detect the two viruses causing mosaic. In ELISA, 12 of 18 sugarcane samples exhibiting varying mosaic symptoms were found to be negative to the virus(es), however they were positive in IC-RT-PCR. We conclude that although DAC-ELISA is simple and cost effective to diagnose these viruses, in samples with very low virus titre, IC-RT-PCR would be more sensitive. This is the first report on the detection of SCSMV and SCMV together in an IC-RT-PCR assay.

Molecular characterization of Indian sugarcane streak mosaic virus isolate

Sugarcane streak mosaic virus (SCSMV), a member of the genus Poacevirus, family Potyviridae, is an important viral pathogen affecting sugarcane cultivation in India. The complete nucleotide sequence of a SCSMV isolate from India (SCSMV-IND) was determined. The linear, assembled, single-stranded positive-sense RNA genome of SCSMV-IND was 9,786 nucleotides in length (excluding the poly (A) tail) and encoded a polyprotein of 3,131 amino acid residues. The genome of SCSMV-IND shared high degree of sequence identity with SCSMV-PAK (93% at nucleotide and 97% at amino acid), and shared only 81% nucleotide and 94% amino acid identities with all the four SCSMV isolates (SCSMV-JP1, -JP2, -ID, and -THA). Phylogenetic tree analysis of the complete genome sequences of SCSMV isolates revealed that they can be clustered into two groups. SCSMV-IND and -AP isolates showed 18% (nucleotide) divergence within the highly conserved 3' partial genome, suggesting a high level of genetic diversity among the Indian SCSMV isolates.

Genetic variability and potential recombination events in the HC-Pro gene of sugarcane streak mosaic virus

Sugarcane streak mosaic virus (SCSMV), a member of the family Potyviridae, is an important viral pathogen affecting sugarcane production in India. The variability in the nucleotide (nt) and amino acid (aa) sequences of helper component proteinase (HC-Pro) of SCSMV isolates from India was investigated and compared with those of previously published virus isolates from different Asian countries. Comparison of all of the sequenced virus isolates revealed a high level of diversity in the HC-Pro gene (72-97% nt sequence identity; 83-99% aa sequence identity), and the Indian isolates were found to be the most divergent (up to 12% variation at the amino acid level). Phylogenetic analysis revealed clustering of 16 SCSMV isolates into two groups. Group I included isolates from India and Pakistan, and group II consisted of isolates from Japan and Indonesia. Recombination analysis revealed nine potentially significant recombination events, and putative recombination sites were identified throughout the HC-Pro gene. Analysis of selection pressure indicated that the HC-Pro gene of SCSMV is under strong negative selection. It is likely that recombination, along with strong negative selection, enhances the speed of elimination of deleterious mutations in the HC-Pro gene.