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Surveys were conducted in 2013 and 2014 to determine geographical variation and phylotypes of RYMV and the influence of environment and climatic factors on their distribution in Morogoro, Pwani, Arusha, Kilimanjaro, Shinyanga, Kigoma, Mbeya and Rukwa regions. Farmers’ field practices and perceptions on the disease were also studied. Most of the farmers (91%) interviewed planted their own saved rice seeds. There were positive correlations (P ≤ 0.05) between weeding methods and source of seeds and occurrence of RYMV disease. The highest prevalence and severity of RYMV were observed in Mbeya and Morogoro regions where mean total rainfall, temperature and RH were 167 and 231 mm, 22.3 and 28°C and 87 and 93%, respectively. The lowest RYMV prevalence was found in Kigoma (9.33%) and Rukwa (11.33%) regions. Results indicated that S4lv, S4lm and S4ug RYMV phylotypes were identified in Arusha, Kigoma, Shinyanga and Kilimanjaro regions for the first time. The strain S5 was still restricted in Kilombero Valley, Morogoro region. Strain S6 was found in new areas (Kigoma and Mbeya regions) where it has not been reported before and new phylotypes of S6 (S6c and S6w) are reported for the first time in this study. New primers specific to RYMV strains (S4, S5 and S6) were also designed in the current study in order to facilitate direct and quick identification of the virus and reduce costly sequencing steps. The RT-PCR amplification products showed that, forward FS5 primer and reverse R20 primer amplified only S5 strains at 278 bp, implying that it is specific for identification of S5 strain. Primers for S4 amplified all S4 and S5 strains at 281 bp, implying that it is not specific, while primers for S6 amplified only S6 strains at 584 bp, implying that it is specific for S6 strains. To avoid the cost of existing methods which require RNA extraction prior to molecular based detection studies, simple, cheap and rapid RYMV
detection methods were optimized. They included Flinders Associates Technology (FTA®) cards, Whatman® paper strips (WPS), nitrocellulose membranes (NCM), immunocapture (IC) and simple-direct-tube (SDT) based on application of RT-PCR. The results indicated that FTA®, WPS, NCM, IC and SDT methods were effective in the preparation, storage and retrieval of viral ribonucleic acids (RNA) from RYMV-infected plant material for direct use in the RT-PCR reactions. However, the longevity of RYMV in FTA and WPS was up to one year at room temperature, while NCM retained longevity of viral proteins for up to 5 days only. Determination of the pathogenic variation of RYMV strains and phylotypes against rice cultivars grown in Tanzania and assessment of resistance-breakdown of the known resistant rice cultivars Azucena (rymv1-1), Gigante (rymv1-2), Tog12387 (rymv1-3), Tog5681 (rymv1-3), Tog5438 (rymv1-4), Tog5672 (rymv1-4 + rymv2) and Tog5674 (rymv1-5) with RYMV1 gene was also done. The rice
cultivars were inoculated with RYMV strains S4lm (Tz526), S4lv (Tz516), S4ug (Tz508), S5 (Tz429, Tz445) and S6c (Tz486) and S6w (Tz539) to evaluate the effect of the RYMV disease on growth characteristics. The results revealed multiple resistance-breaking strains and phylotypes on resistant cultivars Gigante, Tog12387, Tog5438 and Tog5681. Reduction in plant height (2.8%), number of tillers per plant (2.5%), 1 000-grain weight (2.7%), spikelet sterility (3.5%) and reduction in rice yield (5%) were the lowest in rice cultivar Gigante inoculated with strain S6c (Tz486). This study also identified local rice cultivars Kalundi and Mahuhu as resistant to RB RYMV strains and phylotypes (S4lm, S5 and S6w). |
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