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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 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 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 7 th and 12 th 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. |
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