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Not AvailableStudies were conducted to standardize IC-RT-PCR to detect SCSMV and SCMV with more specificity and sensitivity. A total of 30 samples of different varieties of sugarcane which varied in symptoms from severe mosaic symptoms to asymptomatic were diagnosed by DAC-ELISA using recombinant SCSMV coat protein antiserum in which it gave both positive and negative values. Some of the samples with mild symptoms were found to be negative in the assay, hence it warranted further confirmation of negative samples employing molecular tools. In order to accomplish this, a duplex IC-RT-PCR was developed for a simultaneous diagnosis of both the viruses in a single reaction. From DAC-ELISA results, 12 negative samples were diagnosed by duplex IC-RT-PCR using recombinant SCSMV-coat protein antiserum. Among them, eight samples were positive to SCSMV and eleven samples were positive to SCMV, when diagnosed by IC-RT-PCR which indicated that this technique is more sensitive than DAC-ELISA and the results are illustrated by specific bands to SCSMV (~690bp) and SCMV (~380bp) in a single reaction. The antiserum was found to trap both the viruses in IC-RT-PCR. Our assays very clearly indicated that five varieties were positive to SCSMV in both DAC-ELISA and IC-RT-PCR, which confirms that these samples are infected only by SCSMV and not by SCMV. Another four varieties showed mixed infections of the two viruses when diagnosed by duplex IC-RT-PCR. Comparison of IC-RT-PCR with DAC-ELISA revealed that the samples which are negative in DAC-ELISA were positive in IC-RT-PCR and that establishes higher sensitivity of the former technique. Although DAC-ELISA was found to be simple and cost effective to diagnose the virus, in samples with very low titre, IC-RT-PCR would be a more sensitive technique to detect the virus(es). Our study very clearly establishes that IC-RT-PCR is more sensitive to detect the viruses present in low titre more specifically than DAC-ELISA.Not Availabl

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Not AvailableSugarcane wilt caused by Fusarium sacchari was reported one hundred years ago from India and continues to cause significant losses to cane production and productivity in the country. However, research work on this important pathogen was limited especially on characterization of the fungus and its variability. The causal organism was found to vary with time and investigator and the disease could not be reproduced at ease under artificial conditions in the field. During the last 10 years, we made detailed disease surveys in most of the major sugarcane growing regions in the country and collected more than 300 Fusarium isolates infecting the crop. We have established variation in Fusarium isolates associated with sugarcane wilt, based on cultural, morphological, pathogenic and molecular characterization of the isolates. As the phenotype of the pathogen depends on varying environmental conditions, reliability and repeatability of the identity as seen earlier is doubtful. Molecular tools based on DNA analyses are being currently used as an alternative to cultural and morphological characters to characterize the variants of fungal species. Additionally the use of molecular tools for characterization would save time and are more reliable compared to phenotypic characters which vary with time and conditions. Hence further molecular studies were conducted with 50 isolates of the 117 isolates, characterized initially. In all the four different molecular tools viz., sequencing of internally transcribed region (ITS), randomly amplified polymorphic DNA (RAPD), inter-generic sequences-restricted fragment length polymorphism (IGS-RFLP) and inter simple sequence repeats (ISSR) used for characterization, morphologically distinct isolates formed separate clusters and isolates of F. sacchari grouped together in a cluster. Within this cluster, due to intraspecific variation F. sacchari, isolates were further grouped into many subclusters. In all the three different molecular tools used viz., RAPD, IGS-RFLP and ISSR, the chain-forming species separated in a cluster. rDNA ITS sequencing did not separate the F. verticillioides isolates rather it grouped them together with other isolates. However, rDNA ITS sequencing helped in confident prediction of species other than F. sacchari in a separate group. Species other than F. verticillioides viz., F. proliferatum, F. subglutinans and F. napiforme clustered away from F. sacchari. Critical application of the conventional techniques combined with molecular tools clearly established that F. sacchari as the causal agent of wilt in sugarcane. Other Fusarium sp isolated from wilt infected sugarcane stalks were found to be either secondary invaders or non-pathogenic in nature. However, it was found that Fusarium sp associated with pokkah boeng also causes stalk infections and produces wilt in certain varieties. Further studies in this area would bring complete characterization of the Fusaria associated with pokkah beong and wilt in sugarcane. Also epidemiology of these sugarcane diseases will be clearly revealed, especially on survival of F. sacchari and its possible manifestation as foliar as well as stalk disease.Not Availabl

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Not AvailableMosaic 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 assayNot Availabl

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Not AvailableWilt and pokka boeng (PB) caused by Fusarium Sacchari and F. verticilloides in sugarcane are considered to be minor diseases under Coimbatore conditions. However, during the 2009-10 season sudden outbreaks of these diseases appeared in National Hybridization Garden (NHG) where 608 sugarcane hybrid clones are maintained to perform hybridization. Detailed investigations were carried out on the associated pathogens, disease epidemiology, disease resistance in the clones and their management. During 5-6 months stage, initial symptoms of PB were noticed and gradually PB severity increased from chronic to acute phase along with top rot symptoms. As the days progressed affected canes exhibited wilting along with or without top rot symptoms. When the disease severity increased from resistant to susceptible, the affected clones exhibited combined infections of PB and wilt symptoms. Among the different phenotypes of PB and wilt in the clones, top rot and wilt caused more damages in the clones. However, some of the clones exhibited severe wilt alone with more crop loss and severe wilting caused extensive damages to some of the established varieties and parents. Fusaria isolated from the affected tissues were found to be pathogenic in artificial testing. The parental clones in the NHG exhibited a clear variation in their resistance to PB, top rot and wilt and the clones originated from the subtropical region except those from Uchani were found to have more resistant types as compared to those from the tropical states. Although the disease situation was precarious, it was successfully managed by an integrated approach involving chemicals and biocontrol agents in the subsequent seasons. Further studies are in progress to characterize the associated pathogens and their interrelationship in causing PB, top rot and wilt diseases in sugarcane.Not Availabl