Purification and partial characterization of a virus associated with korogwe leaf spot disease of sisal Public Deposited

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  • Sisal Wave sisalana Perrine), one of Tanzania's main cashcrops, is severely affected by korogwe leaf spot disease (KLS).Infected plants show chocolate brown, concentric, scablike eruptions on both surfaces of mature leaves, and produce stained, low-quality fiber. A flexuous, rod-shaped virus was isolated from KLS-infected sisal leaves and studied at Oregon State University. This virus was partially characterized by mechanical transmission, host range,cytopathological effects, electron microscopy, serology anddetermination of some biophysical properties.The virus was sap-transmissible with difficulty to young sisaland common bean. The sisal plants have, however, not shown KLSsymptoms to date. Symptoms induced in beans included severe mosaic,leaf distortion and chlorosis. Occasionally, wilting and deathoccurred. Virus particles were very flexuous, with a helicalsubstructure and about 12.7 nm diameter. They were consistentlyassociated with KLS infection. Tendency for breakage in negativestains, aggregation and adsorption onto plant tissue obscureddetermination of particle modal length. Lengths ranged between 100nm and 4,000 nm with four modes at 950, 1075, 1450 and 1650 nm.Cells of infected sisal and bean leaves contained aggregates ofvirus particles, and characteristic closterovirus inclusions inphloem tissue. None were found in cells of healthy plants.The virus was best purified by gentle grinding with a mortarand pestle, precipitation with Polyethylene glycol (PEG), andrate-zonal centrifugation in sucrose/Cs₂SO₄ cushion gradients. Twobands were formed in these gradients; one at 5.5 cm from the topcontained mainly fragmented particles, and the other at 8.0 cmcontained intact particles, as well as the aggregated ones.The purified virus had a typical closterovirus UV-absorptionspectrum, with A₂₆₀/₂₈₀ ratio of 1.61, and Amax/min ratio of 1.21.In Cs₂SO₄ equilibrium density gradients, the fraction with intactvirus particles had a buoyant density of 1.26 g/cc, while thefraction with predominantly particle fragments had a density of 1.23g/cc.Antiserum prepared against partially purified virus reactedhomologously in microprecipitin tests, immunospecific electronmicroscopy (ISEM) and ELISA. It also reacted to citrus tristezavirus (CTV). Similarly, antisera to CTV produced in Switzerland andFlorida reacted strongly to KLS-associated virus. The two viruseswere however serologically distinguishable in reciprocal ISEM tests.The virus was not serologically related to a virus infecting whitelupines at Oregon State University.In microprecipitin tests, antisera to purified virus reactedhomologously. However, antiserum to the fraction containing virusfragments did not react or reacted weakly with preparationscontaining unbroken particles. Antiserum to the latter reacted withboth virus fractions.KLS-associated virus was detected by ISEM in some symptomlesssisal plants. The produced antiserum should therefore enhancedetection of latent field infections. Previously undetectedvirus-infected plants could be eliminated, thus reducing spread ofthe disease through propagation material. KLS-associated virus isprobably a new Closterovirus. Further characterization of thisvirus is essential for verification.
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