Patrick R. Rubaihayo

Unravelling the genetic diversity of the three main viruses involved in Sweet Potato Virus Disease (SPVD), and its practical implications.

SUMMARY Sweetpotato (Ipomoea batatas) is a widely grown food crop, in which the most important diseases are caused by viruses. Genetic variability of three widely distributed sweetpotato viruses was analysed using data from 46 isolates of Sweet potato feathery mottle virus (SPFMV), 16 isolates of Sweet potato mild mottle virus (SPMMV) and 25 isolates of Sweet potato chlorotic stunt virus (SPCSV), of which 19, seven and six isolates, respectively, are newly characterized. Division of SPFMV into four genetic groups (strains) according to phylogenetic analysis of coat protein (CP) encoding sequences revealed that strain EA contained the East African isolates of SPFMV but none from elsewhere. In contrast, strain RC contained ten isolates from Australia, Africa, Asia and North America. Strain O contained six heterogeneous isolates from Africa, Asia and South America. The seven strain C isolates from Australia, Africa, Asia, and North and South America formed a group that was genetically distant from the other SPFMV strains. SPMMV isolates showed a high level of variability with no discrete strain groupings. SPCSV isolates from East Africa were phylogenetically distant to SPCSV isolates from elsewhere. Only from East Africa were adequate data available for different isolates of the three viruses to estimate the genetic variability of their local populations. The implications of the current sequence information and the need for more such information from most sweetpotato-growing regions of the world are discussed in relation to virus diagnostics and breeding for virus resistance.

Incidence of Viruses and Viruslike Diseases of Sweetpotato in Uganda

Sweetpotato plants were surveyed for viruslike diseases and viruses in the four major agroecological zones of Uganda. Testing of 1,260 sweetpotato plants, of which 634 had virus-like symptoms, showed that virus disease incidence ranged from 2.7% (Soroti district, short grassland-savannah zone) to 20% (Mukono district, tall grass-forest mosaic zone). Sweet potato chlorotic stunt virus (SPCSV), Sweet potato feathery mottle virus (SPFMV), Sweet potato mild mottle virus (SPMMV), and sweet potato chlorotic fleck virus (SPCFV) were serologically detected and positive results confirmed by immunocapture reverse transcriptase polymerase chain reaction (IC-RT-PCR) and subsequent sequence analyses of the amplified fragments, except SPCFV, which lacked sequence information. SPCSV and SPFMV were detected in all the 14 districts surveyed, whereas SPMMV and SPCFV were detected in 13 and 8 districts, respectively. Logistic regression analysis revealed that SPCSV and SPFMV, SPFMV and SPMMV, and SPFMV and SPCFV more frequently occurred together than any other virus combinations or as single virus infections. Co-infections of SPCSV with SPFMV and/or SPMMV were associated with more severe and persistent symptoms than infections with each of the viruses alone. Several plants (11%) displaying viruslike symptoms did not react with the virus antisera used, suggesting that more viruses or viruslike agents are infecting sweetpotatoes in Uganda.

Expression patterns of the gene encoding starch branching enzyme II in the storage roots of cassava (Manihot esculenta Crantz)

Abstract Spatial and temporal expression patterns of the sbeII and sbeI genes, encoding starch branching enzyme II and I, respectively, in cassava (Manihot esculenta Crantz) were studied at different phenological stages of the crop. A partial cDNA for sbeII in cassava was cloned and used along with a cDNA-specific fragment of sbeI. As the cassava plant aged, the transcriptional activity of the sbeII and sbeI genes in the underground storage roots increased, whereas the activity in other organs remained the same or declined. At 180 days after planting (d.a.p.), levels of sbeII and sbeI transcripts in storage roots were very low, whereas at 360 d.a.p., the levels had increased dramatically. The 360 d.a.p. old storage roots also accumulated gbssII and gbssI transcripts, as well as a longer gbssI transcript, gbssI′. The difference between the gbssI and gbssI′ transcripts was shown to be due to differential splicing, whereby the gbssI′ transcript retained the first three introns. Unexpectedly, expression of sbeII and sbeI in the 360 d.a.p. storage roots exhibited fluctuations during the 24 h cycle, both under the normal light/dark regime and under continuous light or continuous dark conditions.

Inheritance of resistance to angular leaf spot in common bean and validation of the utility of resistance linked markers for marker assisted selection out side the mapping population

Inheritance of resistance to angular leaf spot (ALS) disease caused by Phaeoisariopsis griseola (Sacc.) Ferr was investigated in two common bean cultivars, Mexico 54 and BAT 332. Both Andean and Mesoamerican backgrounds were used to determine the stability of the resistance gene in each of the two cultivars. Resistance to P. griseola was phenotypically evaluated by artificial inoculation with one of the most widely distributed pathotypes, 63–39. Evaluation of the parental genotypes, F1, F2 and backcross populations revealed that the resistance to angular leaf spot in the cultivars Mexico 54 and BAT 332 to pathotype 63–39 is controlled by a single dominant gene, when both the Andean and Mesoamerican backgrounds were used. Allelism test showed that ALS resistance in Mexico 54 and BAT 332 to pathotype 63–39 was conditioned by the same resistance locus. Resistant and susceptible segregating populations generated using Mexico 54 resistant parent were selected for DNA extraction and amplification to check for the presence /absence of the SCAR OPN02 and RAPD OPE04 markers linked to the Phg-2 resistance gene. The results indicated that the SCAR OPN02 was not polymorphic in the study populations and therefore of limited application in selecting resistant genotypes in such populations. On the other hand, the RAPD OPE04 marker was observed in all resistant individuals and was absent in those scored susceptible based on virulence data. Use of the RAPD OPE04 marker in marker-assisted selection is underway.

Sugar-mediated semidian oscillation of gene expression in the cassava storage root regulates starch synthesis

Starch branching enzyme (SBE) activity in the cassava storage root exhibited a diurnal fluctuation, dictated by a transcriptional oscillation of the corresponding SBE genes. The peak of SBE activity coincided with the onset of sucrose accumulation in the storage, and we conclude that the oscillatory mechanism keeps the starch synthetic apparatus in the storage root sink in tune with the flux of sucrose from the photosynthetic source. When storage roots were uncoupled from the source, SBE expression could be effectively induced by exogenous sucrose. Turanose, a sucrose isomer that cannot be metabolized by plants, mimicked the effect of sucrose, demonstrating that downstream metabolism of sucrose was not necessary for signal transmission. Also glucose and glucose-1-P induced SBE expression. Interestingly, induction by sucrose, turanose and glucose but not glucose-1-P sustained an overt semidian (12-h) oscillation in SBE expression and was sensitive to the hexokinase (HXK) inhibitor glucosamine. These results suggest a pivotal regulatory role for HXK during starch synthesis. Abscisic acid (ABA) was another potent inducer of SBE expression. Induction by ABA was similar to that of glucose-1-P in that it bypassed the semidian oscillator. Both the sugar and ABA signaling cascades were disrupted by okadaic acid, a protein phosphatase inhibitor. Based on these findings, we propose a model for sugar signaling in regulation of starch synthesis in the cassava storage root.

Sequence variability within the 3 -proximal part of the Sweet potato mild mottle virus genome

Summary. Sweet potato mild mottle virus (SPMMV) is the type member of the genus Ipomovirus (family Potyviridae) and is only known to occur in East Africa. In Uganda, SPMMV is the third most prevalent virus infecting sweet potato. The sequence variability of SPMMV was studied by cloning and sequencing a 1.8-kb fragment representing the 3′-end of the genome of eight SPMMV isolates collected from different districts of Uganda. Sequence comparisons indicated 85.9–99.9% nucleotide sequence identity and 92.8–100% amino acid sequence similarity for the coat protein (CP) encoding region. The nucleotide sequence identity within the 3′-untranslated region (3′ UTR) was 84.7–100%, and the region was variable in length (303–308 nucleotides) due to some deletions within the 5′-proximal part of the 3′ UTR. Phylogenetic analysis of the CP amino acid sequences revealed significant clustering, indicating the existence of distinguishable sequence variants or strains. The low CP amino acid sequence similarity of SPMMV isolates with other characterised viruses of the family Potyviridae and the unusual putative proteolytic cleavage site at the NIb/CP junction further demonstrate SPMMV as a very distinct virus in the family Potyviridae.

Genetic components of pod shattering in soybean

Half diallel crosses among ten pure breeding lines of soybean were made in 1997 and 1998 to study the inheritance of pod shattering in soybean. Evaluation for pod shattering among F2 segregating populations was carried out in an oven set at 80 °C for 12 hours. Diallel analysis was carried out to estimate genetic parameters and detect presence of non allelic interaction of genes affecting pod shattering. Haymans diallel analysis indicated significant variation of Wr + Vr and Wr – Vr over arrays, suggesting epistatic gene action. Similarly results from a joint regression coefficient over replications were significantly (p < 0.05) different from unity and zero, suggesting presence of non allelic interaction of genes. The intercept was positive, suggesting partial dominance for the shattering trait. Both general combining ability (GCA) and specific combining ability (SCA) effects were significant (p < 0.05).

Interactive effects of host, pathogen and mineral nutrition on grey leaf spot epidemics in Uganda

Grey leaf spot incited by Cercospora zeae-maydis is a new devastating foliar disease of maize in East Africa. For effective control, elucidation of the most critical elements of the grey leaf spot disease pyramid is important. This study investigated the role of mineral nutrition, pathogen variability and host resistance in the epidemic. Trials were conducted under field and controlled environments. The 28 isolates used in the controlled environment varied significantly (P ≤ 0.05) in parasitic fitness measured indirectly as disease efficiency, but no infection pattern could be attributed to known C. zeae-maydis pathotypes. Data from field trials showed that host resistance and mineral nutrition significantly (P ≤ 0.05) affected disease efficiency, with highest disease development occurring in nitrogen-augmented plots. Exclusive phosphorus application had no clear effect on grey leaf spot epidemics but combined application with nitrogen significantly (P ≤ 0.05) reduced the predisposition effects of nitrogen to the disease. Overall, treated plots had less disease than unfertilised plots. Fertiliser application had no effect on sporulation capacity, while cultivars significantly affected it. Geographic differences in amount of disease were observed, suggesting environment influences on grey leaf spot incidence. The results suggest that the current grey leaf spot epidemics in East Africa are due to favourable cultivars, poor mineral nutrition and environmental interactions.

Genetic variability and evolutionary implications of RNA silencing suppressor genes in RNA1 of sweet potato chlorotic stunt virus isolates infecting sweetpotato and related wild species.

Background The bipartite single-stranded RNA genome of Sweet potato chlorotic stunt virus (SPCSV, genus Crinivirus; Closteroviridae) encodes a Class 1 RNase III (RNase3), a putative hydrophobic protein (p7) and a 22-kDa protein (p22) from genes located in RNA1. RNase3 and p22 suppress RNA silencing, the basal antiviral defence mechanism in plants. RNase3 is sufficient to render sweetpotato (Ipomoea batatas) virus-susceptible and predisposes it to development of severe diseases following infection with unrelated virus. The incidence, strains and gene content of SPCSV infecting wild plant species have not been studied. Methodology/Principal Findings Thirty SPCSV isolates were characterized from 10 wild Ipomoea species, Hewittia sublobata or Lepistemon owariensis (family Convolvulaceae) in Uganda and compared with 34 local SPCSV isolates infecting sweetpotatoes. All isolates belonged to the East African (EA) strain of SPCSV and contained RNase3 and p7, but p22 was not detected in six isolates. The three genes showed only limited genetic variability and the proteins were under purifying selection. SPCSV isolates lacking p22 synergized with Sweet potato feathery mottle virus (SPFMV, genus potyvirus; Potyviridae) and caused severe symptoms in co-infected sweetpotato plants. One SPCSV isolate enhanced accumulation of SPFMV, but no severe symptoms developed. A new whitefly-transmitted virus (KML33b) encoding an RNase3 homolog (<56% identity to SPCSV RNase3) able to suppresses sense-mediated RNA silencing was detected in I. sinensis. Conclusions/Significance SPCSV isolates infecting wild species and sweetpotato in Uganda were genetically undifferentiated, suggesting inter-species transmission of SPCSV. Most isolates in Uganda contained p22, unlike SPCSV isolates characterized from other countries and continents. Enhanced accumulation of SPFMV and increased disease severity were found to be uncoupled phenotypic outcomes of RNase3-mediated viral synergism in sweetpotato. A second virus encoding an RNase3-like RNA silencing suppressor was detected. Overall, results provided many novel and important insights into evolutionary biology of SPCSV.

Prevalence of viruses infecting cowpea in Uganda and their molecular detection

This item can be retrieved directly from the publishers site at http://dx.doi.org/10.5897/AJB11.398