A reverse transcription-polymerase chain reaction (RT-PCR) assay was shown to be sensitive and reliable for detection of potato mop-top virus (PMTV) RNA sequences in the flesh of infected potato tubers, and in the roots and leaves of soil-bait plants. This assay was compared with an enzyme-linked immunosorbent assay incorporating PMTV specific monoclonal antibodies (TAS-ELISA). The tests were devised to improve the efficiency of detection of viruliferous <I>Spongospora subterranea </I>in agricultural soils, and of PMTV in potato tubers. RT-PCR detected PMTV RNA sequences in the roots and leaves of bait plants after three weeks growth in viruliferous soil, three weeks before the bait plants themselves developed symptoms, and two weeks before virus was detected by TAS-ELISA. Both RT-PCR and TAS-ELISA detected PMTV in the tubers of primary-infected potatoes. RT-PCR and TAS-ELISA were shown to be more sensitive and reliable than conventional bait-tests and sap inoculation methods for the detection and diagnosis of PMTV. PMTV was detected by ELISA in primary zoospores from four out of six isolates of <I>S. subterranea </I>f. sp. <I>subterranea. </I>One virus-free isolate (N) of <I>S. subterranea </I>was used to acquire PMTV from potato roots and to transmit the virus to healthy plants. A mono-fungal culture of <I>S. subterranea </I>(isolate N) was derived by infecting tomato plant roots with a single cystosorus. The culture was used successfully to acquire PMTV from the roots of infected <I>Nicotiana debneyi </I>plants that had been manually inoculated with virus isolates, and subsequently to transmit the virus to test plants. These experiments confirm that <I>S. subterranea </I>is a vector of PMTV. Two PMTV isolates that had been maintained by manual inoculation for 19 and 21 passages were also acquired and transmitted by the fungus culture. A mono-fungal <I>S. subterranea </I>was unable to acquire and transmit PMTV-T which had 700 nucleotides deletion in the coat protein/ read-through (CP/RT) domain, while PMTV-S with a full-length CP/RT gene was efficiently acquired and transmitted by the same fungus culture. Sequence analysis of the 673 nucleotides of the 5'-end of RT gene of PMTV-S indicated that the difference of 700bp in RT gene of PMTV-T relative to that of PMTV-S occurs in 3'-half of the gene and is associated with loss of transmission by the fungus vector, <I>S. subterranea. </I>
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:641054 |
| Date | January 1995 |
| Creators | Arif, Mohammed |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/11343 |
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