Optimization of diagnostic RT-PCR protocols and sampling procedures for the reliable and cost-effective detection of Cassava brown streak virus

Abarshi, M.M.; Mohammed, Ibrahim Umar; Wasswa, Peter; Hillocks, R. J.; Holt, J.; Legg, J.P.; Seal, Susan; Maruthi, M. N.

doi:10.1016/j.jviromet.2009.10.023

Cited by 90 publications

(87 citation statements)

References 18 publications

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“…Using a mixed CBSV and UCBSV infection we identified two cassava elite CBSD infected susceptible varieties usually develop a dry brown-black necrotic rot of the tuberous roots. Despite evidence that CBSVs accumulate in symptomatic and nonsymptomatic root tissues (Abarshi et al, 2010;Moreno et al, 2011), the role of root organs in CBSV replication and cycle has not yet been elucidated. Studies in other plant-virus systems suggest that virus accumulation is not homogenous in root systems and that primary roots can sustain high level of virus replication (Dalmay et al, 2000;Valentine et al, 2002).…”

Section: Discussionmentioning

confidence: 99%

“…Molecular tools available today for detecting and discriminating CBSV species offer new opportunities to better determine the tolerance and resistance levels to CBSVs as well as to investigate cassava response to CBSD (Abarshi et al, 2010;Mbanzibwa et al, 2011a;Moreno et al, 2011;Abarshi et al, 2012;Tomlinson et al, 2013). Importantly, such tools should also be used to investigate the robustness of CBSD resistance when exposed to mixed infections of ipomoviruses and cassava mosaic geminivirus (CMG), which do co-occur in several cassava growing regions (Alicai et al, 2007;Legg et al, 2011).…”

Section: Introductionmentioning

confidence: 99%

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Characterization of Brown Streak Virus–Resistant Cassava

Anjanappa

Mehta

Maruthi

et al. 2016

MPMI

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Characterization of Brown Streak Virus–Resistant Cassava

Anjanappa

Mehta

Maruthi

et al. 2016

MPMI

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“…Several methods exist for the separation of these compounds from DNA, among which is the CTAB method which was first introduced by Taylor and Powell (1982). Since then the method has been widely used for plant DNA extraction often with slight modifications (Lodhi et al, 1994;Barnwell et al, 1998;Michiels et al, 2002;Sharma et al, 2008;Abarshi et al, 2010;Attitalla, 2011;Adeyemi and Ogundipe, 2012;Tiwari et al, 2012).…”

Section: Dna Extractionmentioning

confidence: 99%

Optimised cetyltrimethylammonium bromide (CTAB) DNA extraction method of plant leaf with high polysaccharide and polyphenolic compounds for downstream reliable molecular analyses

Turaki¹,

Ahmad²,

Magaji³

et al. 2017

Afr. J. Biotechnol.

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Yam (Dioscorea species) and banana (Musa species) leaf sample contain high levels of polysaccharide and polyphenolic compounds. Extraction of good quality DNA from such leaves is usually problematic. Therefore, there is a need to extract good quality DNA in order to perform downstream DNA analysis, especially for the detection of viruses due to their low titration in some infected leaves. The cetyltrimethylammonium bromide (CTAB) DNA extraction method was selected in order to optimise DNA extraction from plant material with high level of polysaccharides. Four steps of the Lodhi et al. (1994) method were modified to be user friendly in less resource laboratories of developing countries. Yield and purity of the extracted DNAs were quantified using a NanoDrop 2000 spectrophotometer and by migration on an agarose gel after polymerase chain reaction (PCR) amplification. The results of the DNA yields and purity were in the range of 287.40±2.23 to 424.95±1.85 ng/ul and 2.10±0.05 to 2.19±0.04, respectively. Modification was found to yield DNA of reasonable quantity, quality and purity. Furthermore, the method can be used in a laboratory with less sophisticated waste disposal system.

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“…Healthy cassava plants of the varieties Kaleso (CBSD resistant), Kiroba (tolerant) and Albert (susceptible) were graft-inoculated with either CBSV or UCBSV and infection was confirmed by reverse transcription polymerase chain reaction (RT-PCR; Abarshi et al, 2010Abarshi et al, , 2012Otti et al, 2016). The two viral isolates were collected as stem cuttings from farm fields in Uganda and Mozambique, respectively Maruthi et al, 2014).…”

Section: Cassava Varieties and Virus Isolatesmentioning

confidence: 99%

“…Regenerated plants were grown and scored for CBSD symptoms. Plants that remained symptomless for six months were tested for the presence or absence of CBSV and UCBSV by RT-PCR using virus-specific primers (Abarshi et al, 2010.…”

Section: Effect Of Stem Position and Varieties On Virus Eliminationmentioning

confidence: 99%

Generating virus-free cassava plants by in vitro propagation with chemical and heat treatment

Mohammed¹,

Ghosh²,

Maruthi³

2017

Afr. J. Biotechnol.

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Cassava production in eastern and Central Africa is severely threatened by the current epidemic of cassava brown streak disease (CBSD). The disease is caused by cassava brown streak virus (CBSV)and Ugandan cassava brown streak virus (UCBSV) of the genus Ipomovirus, family Potyviridae. Using virus-free planting material is one effective way to reduce CBSD yield losses in farmer fields. The effects of chemical and heat treatment on tissue cultured plants was investigated to eliminate CBSV and UCBSV infections in three cassava varieties with differing levels of resistance to CBSD. Cassava node buds taken from young shoots of the varieties Kaleso (CBSD resistant), Kiroba (tolerant) and Albert (susceptible) infected with CBSV and UCBSV were grown in vitro in standard Murashige and Skoog tissue culture media, which alone eliminated viruses from up to 30% of the plants. To increase the number of virus-free plants, two-week old virus-infected in vitro plants were exposed to constant temperature regimes of 30, 35, 40 or 45°C for three weeks. Heat treatment at 40°C had the optimum effect as up to 50% virus-free plants were obtained for Kiroba; however, incubation at 45°C was lethal to all three varieties while the lower temperatures produced fewer virus-free plants. Chemical treatment of in vitro plants was performed using the antiviral chemical ribavirin at various concentrations. Ribavirin at 0.10 mM generated up to 40% virus-free plants in all three varieties but concentrations of 0.21 mM were lethal. These findings indicate important methods to produce virus-free planting material; their use by breeders and in the field, will help develop effective control strategies for CBSD management.

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Optimization of diagnostic RT-PCR protocols and sampling procedures for the reliable and cost-effective detection of Cassava brown streak virus

Cited by 90 publications

References 18 publications

Characterization of Brown Streak Virus–Resistant Cassava

Characterization of Brown Streak Virus–Resistant Cassava

Optimised cetyltrimethylammonium bromide (CTAB) DNA extraction method of plant leaf with high polysaccharide and polyphenolic compounds for downstream reliable molecular analyses

Generating virus-free cassava plants by in vitro propagation with chemical and heat treatment

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