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Viruses in Tanzania Beatrice Mwaipopo a Thesis

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Viruses in Tanzania Beatrice Mwaipopo a Thesis 1 INCIDENCE AND MOLECULAR CHARACTERIZATION OF COMMON BEAN (Phaseolus vulgaris L.) VIRUSES IN TANZANIA BEATRICE MWAIPOPO A THESIS SUBMITTED IN FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY OF SOKOINE UNIVERSITY OF AGRICULTURE. MOROGORO, TANZANIA. 2019 2 EXTENDED ABSTRACT Common bean (Phaseolus vulgaris L.) is an important crop grown worldwide. It serves as a main source of protein and starch for over 300 million people in East Africa and Latin America. Despite its importance, production of common bean is constrained by viruses which cause important diseases of common bean. It is also known that different common bean genotypes respond differently to different viruses. In order to ascertain this information, the following specific objectives were established: (1) to characterize common bean viruses isolated from common bean using sequencing molecular techniques, (2) to determine the incidence and distribution of major viruses of common beans in Tanzania, (3) to characterize at molecular level and identify the wild plants harbouring the viruses infecting common beans in Tanzania, (4) to determine the suitable sizes of reads from deep sequenced small RNAs data for VirusDetect software-based detection of common bean viruses using low capability computers, (5) to determine genetic diversity of common bean cultivars and landraces using diversity array technology (DArT) in Tanzania, and (6) to evaluate the response of selected common bean genotypes to four common bean viruses in Tanzania. A total of 7756 common bean samples were collected during survey from five agricultural research zones, while 1340 wild plants samples were collected in four zones except western zone. Total RNAs were extracted using Cetyl trimethyl ammonium bromide method (CTAB). The symptomatic and asymptomatic common bean and wild plant samples were selected and pooled according to their respective zone. Nine and 10 pooled common bean and wild plants, respectively, including the wild plant samples (AIVN-1, AIVN-2 and AIVN-3) that were used in virus mechanical transmission study were sent to Fasteris SA (Switzerland) sequencing company, where small RNAs were sequenced using Illumina HiSeq 2500 platform. However, in wild plants was done on Illumina HiSeq 3000/4000 or Illumina NextSeq platform. Analysis of NGS sequences using VirusDetect Software revealed, 15 viruses, belonging to 11 genera, in the nine pooled common beans RNA samples. Two viruses namely, SBMV and Tomato leaf curl Uganda virus- 3 related Begomovirus were detected for the first time in common bean in Tanzania. In wild plants, NGS detected 122 viruse species in 20 genera. Out of these 122 viruses, 23 viruses from 12 genera were related to viruses known to infect common beans. Peanut mottle virus (PeMoV) and Yam bean mosaic virus (YBMV) were some of the viruses that were detected by RT-PCR in Senna occidentalis and Senna hirsuta, respectively. In mechanical inoculation study, out of 25 symptomatic wild plants samples only four wild plants which belonged to two plant species: Ocimum basilicum L. and Bolusafra bituminosa (L.) Kuntze, were able to infect common beans with Cucumber mosaic virus and a bromovirus closely related Cowpea chlorotic mottle virus, respectively. The wild plants RNA (collected from zones and those used fro mechanical inoculation), were identified by DNA barcoding. However, attempts to sequence 134 PCR products were only successful in only 89 (66.4% success rate). The DNA barcoded plants (89) belonged to 50 plant species. Using RT-PCR, detection of BCMV, BCMNV, CPMMV and SBMV viruses in common bean samples was done. The amplicon were scored to determine the incidence of viruses. Visually assessed field incidence of common bean viral diseases was as high 98%, in Missenyi district. The highest RT-PCR based incidence of BCMV and BCMNV were 36.7% and 76.7%, respectively. The incidence of SBMV ranged from 0 to 90.9%. In northern zone, the highest RT- PCR based SBMV incidence was 10%. The RT-PCR-based CPMMV incidence was highest in eastern zone where the incidence was as high as 46.7%. Also, using primers designed to NGS-based sequences, incidence of five viruses from wild plant (BCMV, BCMNV, CPMMV, YBMV and PeMoV) was determined in 1 430 wild plant samples by RT-PCR. Contrary to NGS results, BCMV, BCMNV and CPMMV were not detected in any wild plant samples. On the other hand, YBMV and PeMoV were detected in three and one wild plant samples, respectively. Genetic diversity of isolates of BCMV, BCMNV and CPMMV from common bean RNA samples was achieved through Sanger sequencing. The obtained nucleotide sequences encoding coat proteins of BCMV, BCMNV and CPMMV isolates revealed they were 90.2 to 100%, 97.1 to 100% and 82.9 to 99.1% similar to 4 each other, respectively. Some isolates, e.g., TZ:Mor 533:2015, had hallmarks of recombination events. In separate study a total of 360 common bean genotypes were grown in screenhouse and DNAs extracted using a CTAB method for genetic diversity analysis using the Diversity array technology (DArT). A total of 35 047 markers were identified of which 558 (1.6%) markers were highly informative. The genetic diversity dendrogram showed that, 278 and 82 common bean genotypes grouped in the Andean and Mesoamerican gene pools, respectively. Principal component analysis (PCA) based on genetic similarity confirmed that the genotypes belonged to two groups (252 genotypes) and their variation was 82.2%. When PCA was determined separately for the Andean and Mesoamerican gene pools, the within similarities were 82.94% and 84.60%, respectively. The response of common bean genotypes to BCMV, BCMNV, SBMV and CPMMV was studied in screen house using a Complete Randomized Design (CRD). Data on disease severity and area under disease progress curve (AUDPC) were subjected to one-way analysis of variance (ANOVA) and post-hoc analysis was done by using Tukey’s test. Depending on the common bean genotype assessed, the symptoms appeared between 7th and 12th days post inoculation for all four viruses. Across all viruses used, disease severity was less than 50% in most common bean genotypes. The AUDPC ranged from 414 – 2 667, 0 – 1 586.7, 105.6 – 1 561.7 and 506 – 2 037 for BCMNV, BCMV, CPMMV and SBMV, respectively. Resistance to all four viruses ranged from susceptible to moderate resistance in inoculated common bean genotypes. However, Fibea and Selian 05 did not develop any symptoms when were inoculated with BCMV (AUDPC = 0). This work represents the first comprehensive surveys of common bean viruses in Tanzania using the of state-of-the-art next generation sequencing technique to simultaneously detect all viruses in common bean samples from five agricultural research zones in Tanzania. Using molecular information, primers were developed, optimized and used to detect viruses – including BCMV, BCMNV, CPMMV, and SBMV – in common bean and wild plants. The incidence of different viruses was determined and the distribution of common bean viruses was mapped. 5 6 DECLARATION I, BEATRICE MWAIPOPO, do hereby declare to the Senate of Sokoine University of Agriculture that this thesis is my own .original work and that it has neither been submitted nor being concurrently submitted for a degree award in any other institution. ……………………. ………………….. Beatrice Mwaipopo Date (PhD Candidate) The above declaration is confirmed by; …………………………… …………………. Prof. Susan Nchimbi-Msolla Date (Supervisor) ………………………… .………………… Dr. P. J. Njau Date (Supervisor) ……………………………… ….……………… Dr. Deusdedith R. Mbanzibwa Date (Supervisor) 7 PUBLICATIONS Mwaipopo, B., Nchimbi-Msolla, S., Njau, P., Tairo, F., William, M., Binagwa, P., Kweka, E., Kilango, M. and Mbanzibwa, D. (2017). Viruses infecting common bean (Phaseolus vulgaris L.) in Tanzania: A review on molecular characterization, detection and disease management options. African Journal of Agricultural Research 12: 1486 - 1500. Mwaipopo, B., Nchimbi Msolla, S., Njau, P., Mark, D. and Mbanzibwa, D. R. (2018). Comprehensive surveys of Bean common mosaic virus and Bean common mosaic necrosis virus and molecular evidence for occurrence of other Phaseolus vulgaris viruses in Tanzania. Plant Disease 102: 2361 - 2370. Nordenstedt, N., Marcenaro, D., Chilagane, D., Mwaipopo, B., Rajamäki, M.-L., Nchimbi-Msolla., S, Njau, P. J. R., Mbanzibwa, D. R. and Valkonen, J. P. T. (2017). Pathogenic seedborne viruses are rare but Phaseolus vulgaris endornaviruses are common in bean varieties grown in Nicaragua and Tanzania. PLoS One 12: e0178242. 8 COPYRIGHT No part of this thesis may be produced, stored in any retrievable system, or transmitted in any form or by any means without a prior written permission of the author or Sokoine University of Agriculture in that behalf. 9 ACKNOWLEDGEMENT I am grateful to the Bill and Melinda Gates Foundation for the financial support through the grant to Dr. Deusdedith Mbanzibwa under the Programme for Emerging Agricultural Research Leader (PEARL; Contract ID OPP1112522). I also acknowledge the Ministry of Agriculture for granting me a four years study leave to undertake my PhD studies. I would like to express my sincere thanks to my supervisors Prof. Susan Nchimbi-Msolla, Dr. P. J. Njau and Dr. Deusdedith Mbanzibwa for supervision, guidance and academic advice that led to the successful completion of this scientific thesis. Special thanks go to the Sokoine University of Agriculture and Department of Crop Science and Horticulture for trusting and admitting me to undertake PhD studies in this reputable agricultural university in the country. I would also like to express my happiness to all members of academic staff of the Department of Crop Science and Horticulture of Sokoine University of Agriculture who played major roles in my study. My special thanks to Tanzania Agricultural Research Insitute (TARI) - Mikocheni for offering me the opportunity to conduct all laboratories works at their institute. My acknowledgement should go to all Research Officers at TARI - Mikocheni, TARI - Uyole, TARI - Maruku, and TARI - Selian who assisted me in one way or another. I specifically thank Ms.
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