Sugarcane bacilliform virus (SCBV), which causes leaf freckle in sugarcane, is a member of the genus Badnavirus. Studies were conducted to characterize SCBV in Saccharum officinarum germplasm and cultivated varieties in India by sequencing the complete genomes of five isolates. Genome lengths ranged from 7,553 to 7,884 nucleotides. Duplications in ORF3 and insertions in the RNase H-domain in some of the isolates were found to contribute to the large size of their genomes. The Indian SCBV isolates share identities of 69-85 % for the complete genomic sequence, indicating wide genetic diversity among them, and share 70-82 % identity with Sugarcane bacilliform Ireng Maleng virus (SCBIMV) and Sugarcane bacilliform Morocco virus (SCBMV), as well as 43-46 % identity with Banana streak virus (BSV) and BSV-related SCBV species from Guadeloupe, indicating that the Indian SCBV isolates are distinct from SCBV isolates reported to date. Irrespective of the region compared, SCBV isolates from India, Australia, and Morocco clustered together. BSV and BSV-related SCBV isolates from Guadeloupe formed another cluster. A phylogenetic analysis based on the partial RT/RNase H-sequence separated SCBV and BSV-related SCBV sequences into 11 SCBV groups viz. SCBV-A to -K. Among the 11 groups, the SCBV sequences separated under H, I, J, and K are newly identified in this study, representing three new species and are tentatively named as SCBBBV, SCBBOV, and SCBBRV. Thus, the PASC and phylogenetic analyses evidenced that the symptoms associated with badnaviruses in sugarcane in India are caused by at least three new species, SCBBBV, SCBBOV, and SCBBRV, besides SCBIMV and SCBMV represented by SCBV-BT and SCBV-Iscam, respectively.

Sixty-three sugarcane leaf samples were collected from fifty-eight sugarcane varieties, evolved from eleven major sugarcane growing states in India, Australia, South Africa and USA. In RT-PCR, using gene specific primers for sugarcane streak mosaic virus (SCSMV)-CP, 58 of 63 sugarcane samples were found positive to the virus infection and rest of the five samples were negative. Partial CP gene sequences of 42 SCSMV isolates including an isolate from aphid colony (Melanaphis indosacchari) infested on sugarcane variety from this study were characterized after cloning and sequencing for selective isolates represented by at least one isolate from each location. The new sequences identified in the study were named as SCSMV-CB isolates. Fifty two sequences including the 10 database sequences (complete CP cds) deposited earlier from this institute were compared with each other as well as GenBank database sequences of Potyviridae members viz., Rymovirus, Potyvirus, Ipomovirus, Tritimovirus and eight sequences of SCSMV reported from elsewhere. Among the SCSMV-CB isolates sequenced in the study, 85.7-100% (nucleotide) and 89.9-100% (amino acid) sequence identities were observed and with the other data base sequences of SCSMV, the respective identities were 82.2-97.5 and 89.7-98.6%. Grouping of the isolates by the maximum likelihood with molecular clock model, distributed 60 SCSMV sequences including the eight database sequences deposited by other SCSMV working groups from India and USA in 16 different phylogenetic groups. Although the isolates of SCSMV were relatively close to Ipomovirus and Tritimovirus, they were sandwiched between Rymovirus and Ipomovirus. The sequence comparison and phylogenetic studies revealed that the relatedness of SCSMV with the potyviral related genera was comparatively low to consider it as a member of earlier described potyviral genera, hence the genus "Susmovirus" (sugarcane streak mosaic virus) has been proposed, with SCSMV as the sole species to be included. The 52 SCSMV-CB isolates from this institute were distributed in 14 phylogenetic groups and the grouping pattern revealed that the virus isolates could not be grouped based on geographical origin of the host varieties or longevity of the host variety.

Sugarcane yellow leaf virus (SCYLV) that causes yellow leaf disease (YLD) in sugarcane (recently reported in India) belongs to Polerovirus. Detailed studies were conducted to characterize the virus based on partial open reading frames (ORFs) 1 and 2 and complete ORFs 3 and 4 sequences in their genome. Reverse-transcriptase polymerase chain reaction (RT-PCR) was performed on 48 sugarcane leaf samples to detect the virus using a specific set of primers. Of the 48 samples, 36 samples (field samples with and without foliar symptoms) including 10 meristem culture derived plants were found to be positive to SCYLV infection. Additionally, an aphid colony collected from symptomatic sugarcane in the field was also found to be SCYLV positive. The amplicons from 22 samples were cloned, sequenced and acronymed as SCYLV-CB isolates. The nucleotide (nt) and amino acid (aa) sequence comparison showed a significant variation between SCYLV-CB and the database sequences at nt (3.7-5.1%) and aa (3.2-5.3%) sequence level in the CP coding region. However, the database sequences comprising isolates of three reported genotypes, viz., BRA, PER and REU, were observed with least nt and aa sequence dissimilarities (0.0-1.6%). The phylogenetic analyses of the overlapping ORFs (ORF 3 and ORF 4) of SCYLV encoding CP and MP determined in this study and additional sequences of 26 other isolates including an Indian isolate (SCYLV-IND) available from GenBank were distributed in four phylogenetic clusters. The SCYLV-CB isolates from this study lineated in two clusters (C1 and C2) and all the other isolates from the worldwide locations into another two clusters (C3 and C4). The sequence variation of the isolates in this study with the database isolates, even in the least variable region of the SCYLV genome, showed that the population existing in India is significantly different from rest of the world. Further, comparison of partial sequences encoding for ORFs 1 and 2 revealed that YLD in sugarcane in India is caused by at least three genotypes, viz., CUB, IND and BRA-PER, of which a majority of the samples were found infected with Cuban genotype (CUB) and lesser by IND and BRA-PER genotypes. The genotype IND was identified as a new genotype from this study, and this was found to have significant variation with the reported genotypes.

No abstract

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.

Sugarcane mosaic virus (SCMV) is one of the two causative viruses of mosaic in sugarcane, a sugar crop widely grown under tropical and subtropical conditions worldwide. Although molecular characterization of SCMV strains was reported from many countries, strains occurring in India, a major sugarcane producer have not been reported so far. Twenty-six sugarcane samples represented by seven major sugarcane growing states in India and USA were subjected to reverse-transcription polymerase chain reaction (RT-PCR) using a pair of newly designed coat protein specific primers. Among them 17 were found positive to the SCMV infection. The lengths of the sequences derived in this study using the new set of primers varied between 812 and 866 nt. The amino acid sequence comparison of 30 Indian SCMV isolates showed wide range of sequence similarities in core region (88.80-100%) and hyper variable region (51.3-100%). In the N-terminal region of the five Indian isolates, a deletion of 12 aa residues between aa 11 and 30 was observed, whereas the deletion was between aa 45 and 50 in SCMV-B and -D and between aa 61 and 70 in SCMV-A. The phylogenetic analyses performed with 46 SCMV CP sequences for both hyper variable region and core region separated the isolates mostly according to their geographical origin. The 30 Indian SCMV isolates were included exclusively in four groups besides SCMV-IND, which was grouped with SCMV-SC, a type of strain from Australia. Nearly 97.0% of the Indian isolates have no signs for close relationships with previously characterized SCMV type strains (SCMV-A, -B, -D, -E, and -SC) reported from other countries. Our studies revealed that the sugarcane mosaic in India are caused by at least nine new SCMV variants (IND-CC1, -CC2, -CC3, -CC4, -CO1, -CO2, -CP, -CS, and -J) and a type strain SCMV-SC represented by SCMV-IND. This is the first report on the variability and occurrence of new SCMV population in India.

Sugarcane grassy shoot (SCGS) caused by SCGS-phytoplasmas is a serious disease of sugarcane. Since the diseased plants exhibit various phenotypic symptoms under field conditions, diagnosis of the disease becomes difficult. A detailed study was conducted on validating phytoplasma specific primers for the diagnosis of the disease and to identify the extent of variations in rDNA genome of SCGS-phytoplasmas. A total of 37 sugarcane samples with different phenotypic symptoms of SCGS were collected from various states of India. Nested-PCR amplification using two sets of forward (P1/P7) and reverse primers (P4/P7) of 16S rRNA gene, 16S-23S rRNA spacer region (SR) and 23S rRNA gene sequences amplified SCGS-phytoplasmas in all the suspected samples. Nucleotide sequence analysis clearly revealed that 18 isolates of SCGS-phytoplasmas from different locations/ varieties showed no variation in the targeted gene sequences. Although there were significant variations in phenotypic expression of SCGS phytoplasmas on sugarcane, we could not establish any genotypic variation among the pathogenic isolates in rDNA region. All our sequences matched exactly with other reported phytoplasmal isolates of SCGS-Pun (DQ459439), SCGS (DQ380341), SCGS-CM1 (AM269740) and SCGS-SV1 (AM269742) from India. Among the other reported SCGS phytoplasmas it shared sequence similarity ranging from 99.7 to 94.3%. Our SCGS phytoplasmas showed 99.6% similarity with other phytoplasmas infecting sugarcane such as sugarcane white leaf (SCWL) and 97.8% with SCYL. The closely related phytoplasmas such as BraWL and SGS phytoplasmas showed 99.6% similarities with our SCGSphytoplasmas. However, distantly related phytoplasmas FBP and CBY1 and StLL showed sequence similarities between 84.3 and 82.4%. Restriction of the amplicons with a set of restriction enzymes did not show any polymorphism among them. 16S rRNA, 16S-23S rRNA SR and 23S rRNA sequencing and restriction fragment length polymorphism (RFLP) results proved that the phytoplasma isolates infecting sugarcane in India with phenotypic variations from different regions have similar ITS region and further studies are needed to locate other genes in phytoplasma genome which determine variations in phenotypic expression in the SCGS disease.

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.