Characterization of alectra vogelii (witch weed) strains using molecular markers in selected parts of Malawi and Tanzania

Alectra vogelii has been the major constraint known to attack leguminous species especially to cowpea production. Identification of genetic variation of A.vogelii is a pre-requisite for developing improved cowpea varieties. Hence, the objective of the experiment was the identification of phylogenetically differences and differential responses of A. vogelii found in cowpeas, bambara groundnuts and sunflower from selected parts of Malawi and Tanzania.The first objective, total of 240 SSRs (Rice bean, S. gesnerioides and S. hermothica markers), ISSRs, cpDNA and mtDNA primers used to determine the genetic variability of A.vogelii. The PCR master mix reaction volume of 25μl, containing 2.5μl of 10X PCR buffer, sterile distilled water, 1μM of each primer, 1mM of each dNTPs, 0.5U/μl of TaqDNA polymerase and 50 ng DNA. PCR and gel electrophoresis ran. High coefficients of genetic similarity were revelled among A.vogelii variants. The 29 A.vogelii isolates examined was clustered into six main groups. The identified genetic variability of the A.vogelii will help in effective breeding of sunflower, bambara groundnuts and groundnuts. Second objective, the 23 isolates of A. vogelii was infested to 11 cowpea varieties/lines (Vuli 1, Vuli 2, Fahari, Tumaini, IT99K-573-1, IT99K-7-21-2-2-1, IT00K-1263, IT99K-1122, IT82K-16, B301, Bunda 1, and TZA 263), made a total of 492 pots. Approximately 500 A. vogelii seeds were infested in the prepared pots, 3 seeds of cowpea were sown approximately 5cm deep. The number of emerged A. vogelii plants was counted. The results show that, there is high variability in cowpeas depending on number of the parasite emergence and time of emergence. Third objective, a total of 21 A.vogelii isolate was infested to Bambara groundnuts, peanuts and soyabeans, which made the total of 126 pots. The plants were watered after every 3 days. The number of emerged A. vogelii plants was counted at 6th, 8th, 10th and 12thweeks after pot infestation. Both A.vogelii isolates from Tanzania and Malawi showed late emergence and were more reactive on bambara groundnuts than on soyabean and groundnuts

Characterization of alectra vogelii (witch weed) strains using molecular markers in selected parts of Malawi and Tanzania

Alectra vogelii has been the major constraint known to attack leguminous species especially to cowpea production. Identification of genetic variation of A.vogelii is a pre-requisite for developing improved cowpea varieties. Hence, the objective of the experiment was the identification of phylogenetically differences and differential responses of A. vogelii found in cowpeas, bambara groundnuts and sunflower from selected parts of Malawi and Tanzania.The first objective, total of 240 SSRs (Rice bean, S. gesnerioides and S. hermothica markers), ISSRs, cpDNA and mtDNA primers used to determine the genetic variability of A.vogelii. The PCR master mix reaction volume of 25μl, containing 2.5μl of 10X PCR buffer, sterile distilled water, 1μM of each primer, 1mM of each dNTPs, 0.5U/μl of TaqDNA polymerase and 50 ng DNA. PCR and gel electrophoresis ran. High coefficients of genetic similarity were revelled among A.vogelii variants. The 29 A.vogelii isolates examined was clustered into six main groups. The identified genetic variability of the A.vogelii will help in effective breeding of sunflower, bambara groundnuts and groundnuts. Second objective, the 23 isolates of A. vogelii was infested to 11 cowpea varieties/lines (Vuli 1, Vuli 2, Fahari, Tumaini, IT99K-573-1, IT99K-7-21-2-2-1, IT00K-1263, IT99K-1122, IT82K-16, B301, Bunda 1, and TZA 263), made a total of 492 pots. Approximately 500 A. vogelii seeds were infested in the prepared pots, 3 seeds of cowpea were sown approximately 5cm deep. The number of emerged A. vogelii plants was counted. The results show that, there is high variability in cowpeas depending on number of the parasite emergence and time of emergence. Third objective, a total of 21 A.vogelii isolate was infested to Bambara groundnuts, peanuts and soyabeans, which made the total of 126 pots. The plants were watered after every 3 days. The number of emerged A. vogelii plants was counted at 6th, 8th, 10th and 12thweeks after pot infestation. Both A.vogelii isolates from Tanzania and Malawi showed late emergence and were more reactive on bambara groundnuts than on soyabean and groundnuts

Characterization of alectra vogelii (witch weed) strains using molecular markers in selected parts of Malawi and Tanzania

Master DisertationAlectra vogelii has been the major constraint known to attack leguminous species especially to cowpea production. Identification of genetic variation of A.vogelii is a pre-requisite for developing improved cowpea varieties. Hence, the objective of the experiment was the identification of phylogenetically differences and differential responses of A. vogelii found in cowpeas, bambara groundnuts and sunflower from selected parts of Malawi and Tanzania.The first objective, total of 240 SSRs (Rice bean, S. gesnerioides and S. hermothica markers), ISSRs, cpDNA and mtDNA primers used to determine the genetic variability of A.vogelii. The PCR master mix reaction volume of 25μl, containing 2.5μl of 10X PCR buffer, sterile distilled water, 1μM of each primer, 1mM of each dNTPs, 0.5U/μl of TaqDNA polymerase and 50 ng DNA. PCR and gel electrophoresis ran. High coefficients of genetic similarity were revelled among A.vogelii variants. The 29 A.vogelii isolates examined was clustered into six main groups. The identified genetic variability of the A.vogelii will help in effective breeding of sunflower, bambara groundnuts and groundnuts. Second objective, the 23 isolates of A. vogelii was infested to 11 cowpea varieties/lines (Vuli 1, Vuli 2, Fahari, Tumaini, IT99K-573-1, IT99K-7-21-2-2-1, IT00K-1263, IT99K-1122, IT82K-16, B301, Bunda 1, and TZA 263), made a total of 492 pots. Approximately 500 A. vogelii seeds were infested in the prepared pots, 3 seeds of cowpea were sown approximately 5cm deep. The number of emerged A. vogelii plants was counted. The results show that, there is high variability in cowpeas depending on number of the parasite emergence and time of emergence. Third objective, a total of 21 A.vogelii isolate was infested to Bambara groundnuts, peanuts and soyabeans, which made the total of 126 pots. The plants were watered after every 3 days. The number of emerged A. vogelii plants was counted at 6th, 8th, 10th and 12thweeks after pot infestation. Both A.vogelii isolates from Tanzania and Malawi showed late emergence and were more reactive on bambara groundnuts than on soyabean and groundnuts

Next-generation sequencing-based detection of common bean viruses in wild plants from tanzania and their mechanical transmission to common bean plants

Publisher Copyright: Copyright © 2021 The Author(s).Viral diseases are a major threat for common bean production. According to recent surveys, >15 different viruses belonging to 11 genera were shown to infect common bean (Phaseolus vulgaris L.) in Tanzania. Virus management requires an understanding of how viruses survive from one season to the next. During this study, we explored the possibility that alternative host plants have a central role in the survival of common bean viruses. We used next-generation sequencing (NGS) techniques to sequence virus-derived small interfering RNAs together with conventional reverse-transcription PCRs (RT-PCRs) to detect viruses in wild plants. Leaf samples for RNA extraction and NGS were collected from 1,430 wild plants around and within common bean fields in four agricultural zones in Tanzania. At least partial genome sequences of viruses potentially belonging to 25 genera were detected. The greatest virus diversity was detected in the eastern and northern zones, whereas wild plants in the Lake zone and especially in the southern highlands zone showed only a few viruses. The RT-PCR analysis of all collected plant samples confirmed the presence of yam bean mosaic virus and peanut mottle virus in wild legume plants. Of all viruses detected, only two viruses, cucumber mosaic virus and a novel bromovirus related to cowpea chlorotic mottle virus and brome mosaic virus, were mechanically transmitted from wild plants to common bean plants. The data generated during this study are crucial for the development of viral disease management strategies and predicting crop viral disease outbreaks in different agricultural regions in Tanzania and beyond.Peer reviewe

Comprehensive survey of common bean viruses in Tanzania using next generation and Sanger sequencing techniques

<p>Common bean (<em>Phaseolus vulgaris</em> L.) is an important legume crop in Tanzania and elsewhere in the tropics and subtropics. We employed a next-generation sequencing technique to detect viruses in common bean plant samples collected from five agricultural research zones in the country. The aim was to target and sequence virus-derived small RNAs. To achieve this, total RNA was isolated from dry leaf samples using the CTAB method. The CTAB buffer contained 2% CTAB, 100 mM Tris–HCl, 20 mM EDTA, 2.5 M NaCl, freshly prepared 1% sodium sulfite, 2% PVP and 2.5% 2-mercaptoethanol in nuclease-free water. Total RNA was sent to Fasteris in Switzerland where the small RNA was purified by electrophoresis in an acrylamide gel. The small RNA library was prepared using the Illumina TrueSeq small RNA  sample preparation kit (Illumina Inc., San Diego, CA, USA). Viruses were detected using VirusDetect software (v.1.6 and v.1.7) (available at http://bioinfo.bti.cornell.edu/cgi-bin/virusdetect/index.cgi) and supercomputer at CSC.fi. Viruses detected belonged to at least 11 genera.</p

Detection of PvEV-1 by RT-PCR in common beans in Tanzania.

<p>In the list below, landraces are marked with asterisk (*). Other samples represent improved varieties (origin of samples shown in parenthesis). Lane labelled ‘M’ represents a O'GeneRuler 1 kb Plus DNA ladder. The expected size of PCR products was 374 bp. Lanes 1, ‘Njugu’* (Southern Highlands zone); 2, ‘pooled RNA’ (Southern Highlands zone); 3, ‘pooled RNA’ (Eastern zone); 4, ‘pooled RNA’ (Northern zone); 5, ‘Rosekoko’/’Lyamungu 85’ (Eastern zone); 6, ‘Salundi’ (Southern Highlands zone); 7, ‘E 36’ (Southern Highlands zone); 8, ‘Msafiri’* (Southern Highlands zone); 9, ‘Msafiri’* (Eastern zone); and 10, ‘Mshindi’ (Eastern zone).</p

Conserved domains in the polyprotein encoded by PvEV-1 and PvEV-2 from Nicaragua.

<p>Numbers indicate the residues defining the conserved domains. Hel-1, helicase; CPS, putative capsular polysaccharide synthase; UGT, UDP-glycosyltransferase; RdRp, RNA-dependant RNA polymerase; and MTR, methyltransferase.</p

Viruses detected in the pools of common bean samples from Nicaragua (NI) and Tanzania (TZ) by VirusDetect.

<p>Viruses detected in the pools of common bean samples from Nicaragua (NI) and Tanzania (TZ) by VirusDetect.</p

Symptoms observed in common bean plants in La Compañia, Nicaragua.

<p>(a), Stunting of the plant, malformation and blistering of leaves. (b), Mild epinasty and vein reversion. (c), Green-yellow chlorosis. (d), Green-yellow mosaic.</p

Identification of PvEV-2 in the sample pool HXH8 from the Southern Highland zone of Tanzania based on small-RNA deep sequencing.

<p>(a), Viral contigs (red bars) mapped to the sequence of PvEV-2-Okada (AB719398) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0178242#pone.0178242.ref025" target="_blank">25</a>] using VirusDetect [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0178242#pone.0178242.ref051" target="_blank">51</a>]. Each nucleotide in the contigs was covered by siRNA reads at least 5 times. (b) The 21- to 24-nt reads mapped to the sequence of PvEV-2. The <i>x</i> axis and the scale below the figure depict the viral genome and nucleotide positions, respectively. The <i>y</i> axis indicates the number of siRNA reads derived from the coding strand (blue bars above the <i>x</i> axis) and complementary strand (red bars below the <i>x</i> axis).</p