Potato is one of the most important crops grown in Canada and all over the world. Late blight caused by P. infestans is one of the major diseases of potato and is mainly managed by fungicides application. The extensive use of fungicides not only causes adverse effects on the environment but also accelerates the development of resistance in this pathogen. Horizontal resistance is considered as the best choice to control P. infestans as it is durable over years. Breeding for durable resistance requires evaluation of hundreds of breeding lines in greenhouses and in the field. This is usually done by testing several epidemiological parameters such as infection efficiency, lesion size, latent period, and area under disease progress curve (AUDPC). These methods are time-consuming and expensive. The present study reports standardization of metabolic profiling protocols and exploration of metabolic profiling based on GC/MS as an additional tool to discriminate resistance in potato against late blight. Potato cultivars varying in horizontal resistance against late blight have been inoculated with water or the pathogen and more than 100 metabolites have been tentatively identified by GC/MS. Univariate analysis has been used to identify several pathogenesis related (PR) and defense related (DR) metabolites that have potential for application as resistance biomarker metabolites. Multivariate analysis of the abundances of metabolites (the mass spectral (MS) ion trap detector outputs were obtained using Saturn Lab Software Version 5.52 and these abundances are positively proportional to the concentration of mass ions of metabolites) in cultivars were mainly used to identify pathogenesis and resistance functions. Following pathogen inoculation, several metabolites such as amino acids, organic acids, fatty acids and sugars, were significantly increased in abundances, especially in the resistant cultivar. Other metabolites such as phenylalanine, tyrosine, shikimic acid and malonic acid detected here are well known for their direct participation in the shikimic acid, the phenylpropanoid, and the malonic acid metabolic pathways. These pathways lead to the production of several defense metabolites including antimicrobial compounds including phenolics, flavonoids and phytoalexins. The metabolic profiling technology developed here has the potential application for screening of potato breeding lines for horizontal resistance against late blight.

Complementary DNA (cDNA) clones representing approximately 5800 nucleotides of potato leafroll virus (PLRV) genomic RNA were generated, restriction-mapped, and partially sequenced. Within one of the cDNA clones an open reading frame (ORF) that could encode a 23 kDa protein was identified and further characterized. Comparison of the deduced amino acid sequence with the coat protein amino acid sequence of the PAV strain of barley yellow dwarf luteovirus (BYDV-PAV) showed significant similarity. This observation together with its size and internal location within the genome suggested that this gene encoded the PLRV coat protein. Other similarities were observed between PLRV and BYDV sequences in this region of their genomes, including a 17 kDa ORF within the ORF encoding the 23 kDa coat protein, and termination of the latter with an amber codon which is immediately followed by a large ORF in the same reading frame. Three PLRV coat protein gene sequences were used to transform tobacco and the potato cultivars 'Desiree' and 'Russet Burbank' via Agrobacterium tumefaciens mediated gene transfers. One construct possessed 12 nucleotides of the untranslated leader sequence 5' to the putative coat protein gene start codon. The other construct, which was also inserted in the reverse orientation to produce negative-sense RNA, had 192 nucleotides from this leader sequence. When these sequences were introduced as chimaeric genes under the control of a duplicated cauliflower mosaic virus 35S (CaMV) promoter, transcription levels were high. Both positive-sense transcripts produced potato leafroll coat protein which accumulated to maximum levels of approximately 0.5% and 0.01% of total leaf protein in tobacco and potato, respectively. Results show that significant levels of inoculum concentration-independent sustained resistance were obtained with each construct, resulting in PLRV titres as low as 1% of the level observed in untransformed plants, as determined by enzyme-linked immunosorbent assays. Virus transmission from PLRV-inoculated transgenic 'Russet Burbank' was reduced substantially and was correlated with virus titre. The pattern and level of protection were the same for constructs producing positive- and negative-sense RNA, suggesting a similar mechanism of resistance. Virus levels were negatively correlated to transcript levels within the transgenic plants. This resistance will have practical applications for the control, of PLRV. Elucidation of the mechanism of resistance may also help understand the mechanisms of virus infection. / Land and Food Systems, Faculty of / Graduate

No description available.

Call number: LD2668 .T4 1960 P65

Call number: LD2668 .T4 1985 L33 / Master of Science

The sequence of 3553 nucleotides corresponding to the 3'-terminal region of potato virus S (PVS) has been determined from cloned cDNA. The sequence obtained contains six open reading frames with the potential to encode proteins of Mr 10,734, Mr 32,515, Mr 7,222, Mr 11,802, Mr 25,092 and at least Mr 41,052. The amino acid sequence of the 33K ORF has been confirmed to be that of the viral coat protein gene. The nucleotide sequence of this ORF was obtained from expression plasmids which were isolated by binding with a specific monoclonal antibody to PVS, and the expression of coat protein fusion products was verified by Western blots of bacterial cell lysates. The deduced amino acid sequence of a 70 amino acid portion from the central region of the PVS coat protein was 59% identical to the analogous region of potato virus X. In addition, the 7K, 12K and 25K ORF's displayed significant sequence homology with similar sized ORF's from a number of potexviruses. The partial 41K ORF was homologous with the C-terminal portion of the viral replicase proteins of potato virus X and white clover mosaic virus. While the biological functions of the 12K and 25K non-structural proteins coded for by PVS and members of the potexvirus group remain unknown, the 12K protein displays a hydropathicity profile consistent with a membrane associated protein and the 25K protein contains a conserved sequence motif found in a number of nucleoside triphosphate binding proteins. Members of the carlavirus group are distinguished from the potexviruses by the presence of a small [11K (PVS, potato virus M) - 16K (lily symptomless virus)] 3' terminal ORF which appears to contain a sequence motif similar to the 'zinc-finger' domain found in many nucleic acid binding proteins. The coat protein gene from potato virus S (PVS) was introduced into Nicotiana debneyii tobacco as well as a commercial potato cultivar, 'Russet Burbank', by leaf disc transformation using Agrobacterium tumefaciens. Transgenic plants expressing the viral coat protein were highly resistant to subsequent infection following mechanical inoculation with the Andean or ME strains of PVS as indicated by a lack of accumulation of virus in the upper leaves. The coat protein mediated protection afforded by these transgenic plants was sufficient to prevent the accumulation of virus in the tissues of non-transformed 'Russet Burbank' shoots which had been grafted onto transgenic plants inoculated with PVS, and in reciprocal grafts, transgenic shoots accumulated less than 2% (6 weeks after grafting) of the concentration of PVS found in non-transformed shoots similarly grafted onto plants systemically infected with PVS. These transgenic plants also displayed a measure of resistance to inoculation with a related carlavirus from potato, potato virus M. In agreement with previous reports for plants expressing PVX coat protein, plants expressing PVS coat protein were also protected from inoculation with PVS RNA. These results provide further evidence that coat protein mediated protection for these two groups of viruses, which share similar genome organizations, may involve inhibition of some early event in infection, other than, or in addition to, virus uncoating. Specific monoclonal antibodies were prepared against a C-terminal derived 18 kDa portion of the 25K protein of PVS expressed as an in-frame chimeric fusion protein with the glutathione S-transferase gene. The in vivo expression of this non-structural protein in virus infected tissue, as well as tissue from transgenic tobacco (var Xanthi-nc) engineered to contain the entire 25K gene, was verified by Western immunoblot labelling. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

Polygalacturonase-inhibiting proteins (PGIPs) are plant proteins believed to playa role in the defence against pathogenic fungi. In this study. it was hypothesized that apple PGIPI could be used to confer enhanced resistance against Verticillium-wilt. a major disease of potato caused by the fungus Verticillillm dahliae. Transgenic lines containing the apple pgip1 gene under control of the enhanced CaMV 35S (e35S) promoter had been generated previously. Stable integration of the transgene into the potato genome was shown by the polymerase chain reaction (PCR) and Southern blot with a DIG¬labelled apple pgip1 fragment as probe. Polygalacturonase (PG)-inhibiting assays (the agarose diffusion assay and reducing sugar assays) were employed to investigate the inhibiting activity of apple PGIP I extracts, prepared from the transgenic potato lines. on the PGs secreted by V. dahliae grown on pectin medium. Inhibition was successful for all but one of the transgenic lines. Active PGIPI was expressed in the leaves of in vitro- and glasshouse grown plants, as well as in roots of in vitro-grown plants. Due to the success of the in vitro inhibition results. it was anticipated that the apple pgip1 transgene would protect the transgenic lines against Verticillium-wilt in a subsequent glasshouse trial. The transgenic lines and untransformed BP I potato control were planted in soil inoculated with V. dahliae microsclerotia and control soil. Assessments of the visual symptoms of yellowing and wilt were made on a scale of 1-5. Colonisation of stem sections was determined by plating onto potato dextrose agar plates. Disease index values were calculated from the symptom and colonisation data. Analysis of variance indicated six lines to be significantly different from the rest when grown in the inoculated soil, but five of them also showed significantly slower senescence symptoms when grown in the control soil. It is proposed that the physiological effect of an extended juvenile phase resulted in the apparent increased disease resistance. This could be caused by transformation or tissue culture¬-induced somaclonal variation of the potato plants. The hypothesis that transformation of the apple pgip1 gene into potato would confer enhanced resistance against Verticillium-wilt was not supported by the data that was obtained. Expression of antifungal genes by pathogen-inducible promoters is a valuable strategy in the development of disease resistant crops of importance. A construct containing the apple pgipl gene under control of the pathogen-inducible gst1 promoter from Arabidopsis thaliana (L.) Heynh was generated. Agrobacterium tumefaciens GV31OI(pMP90RK) was transfonned with the plant transformation vector pCAMBIA2300 containing the gst1 and e35S promoter-pgip1 inserts. A. thaliana was transformed using the floral-dip method, and putative transgenic progeny were selected by kanamycin selection of the seeds. PCR verified the insertion of the transgene into the genomes of T2 and T3 lines. Gene expression from the two promoters was compared by performing PGIP extractions and the agarose diffusion assay. The gst1 promoter was active even without induction by methyl-salicylate. Both constructs led to the expression of active apple PGIP1 against V. dahliae PG in the heterologous plant A. thaliana. / Dissertation (MSc (Plant Biotechnology))--University of Pretoria, 2006. / Plant Science / unrestricted

Alternaria leaf petiole and stem blight is an important disease of sweetpotato (Ipomoea batatas (L.) Lam.) causing yield losses in both landraces and improved cultivars. The most important species causing economic yield loss in Uganda are Alternaria bataticola and A. alternate with A. bataticola the most aggressive and widely distributed. The study was conducted to: i) establish farmer-preferred sweet potato attributes, production constraints and Alternaria leaf petiole and stem blight awareness; ii) evaluate Ugandan sweet potato germplasm for Alternaria leaf petiole and stem blight resistance; iii) determine the mode of inheritance of resistance to Alternaria leaf petiole and stem blight and storage root yield components of sweet potato through estimation of the general combining ability (GCA) of the parents and the specific combining ability (SCA) of the parents for each cross; and iv) determine the adaptability and farmer acceptability of selected F1 genotypes across environments. The participatory rural appraisal was conducted to establish farmer preferences and production constraints revealed that farmer preferred sweet-potato traits were high yield, sweetness (taste), early maturity, high dry mass, resistance to pests and diseases, and in-field root storability after maturity. A majority of the farmers considered Alternaria leaf petiole and stem blight a serious production constraint causing yield loss of over 50%. The main control measures against the disease were roguing of infected plants, spraying with fungicides, use of healthy planting materials and planting resistant genotypes. Thirty sweet potato land races and improved cultivars were evaluated for Alternaria blight severity; yield, dry mass, harvest index, sweetpotato weevil (Cylas spp.) damage and sweetpotato virus disease at two sites (Namulonge and Kachwekano) over three seasons (2010B, 2011A, 2011B) under Alternaria inoculum and fungicide spray treatments. Landrace Shock was more resistant to Alternaria blight than Tanzania, the resistant check. Genotypes NASPOT 1, NASPOT 7, New Kawogo and Dimbuka were the most susceptible. Thirty two F1 families were generated from 16 parents in two sets in a North Carolina II mating scheme. The families were evaluated at two sites using a 5 x 7 row-column design with two replications. There were significant (P<0.05) differences among the families in Alternaria blight severity. Both GCA and SCA mean squares (MS) for Alternaria blight were highly significant (P<0.001) but the predominance of GCA sum of squares (SS) for Alternaria blight at 67.4% of the treatment SS versus 32.6% for SCA SS indicated that additive effects were more important than the non-additive effects in controlling this trait. For the yield components, the GCA MS were significant (P<0.05) and accounted for more than 60% of the treatment SS except for percentage dry mass composition where SCA SS accounted for 53.0% of the treatment SS implying that non-additive genetic effects were slightly more important than additive for this trait. Some parents that had desirable high, negative GCA effects for Alternaria blight produced families with undesirable positive SCA effects and the reverse was also true. This implied that the best parents should not be chosen based on GCA effects alone but also on SCA effects of their best crosses. The promising F1 genotypes selected from previously evaluated crosses together with one Alternaria blight resistant check (Tanzania) and one susceptible check (NASPOT 1) were evaluated at three sites (Namulonge, Kachwekano and Serere) using a randomised complete block design with three replications. Scientists and farmers evaluated the agronomic performance and also quality traits of the genotypes before and at harvest. Genotypes G14, G16, G24, G29, G49, G59 and G69 were the most stable across the sites for low Alternaria blight severity and can, therefore, be recommended for further evaluation under both low and high disease pressure areas. Genotypes G67, G13, G14, G24, G29 and G53 were the most high yielding and stable across the sites and were therefore the most widely adapted. In the participatory selection, before harvest and at harvest, Spearman’s rank correlation of the scientists and farmers’ mean ranking of the genotypes at each site was positive and significant. This indicated that the scientists in the study were capable of selecting for farmer preferred traits. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2012.

A three-year (1998-2000) field and laboratory study conducted in Fredericton, New Brunswick, Canada analyzed and quantified the dispersal of adult Colorado potato beetles within and between conventional and transgenic potato plots established according to the high-dose/refuge strategy. More specifically, the study addressed the following four predictions: (1) Adult Colorado potato beetle abundance or flight activity in transgenic potato fields is positively correlated to the abundance or flight activity in the immediately adjacent refuge field. (2) Colorado potato beetle intraspecific competition on potato plants will increase the flight take-off frequency of adult CPB; tolerating relatively high numbers of CPB egg masses or larvae or a high level of CPB defoliation on potato plants in the refuge could therefore be considered to increase the movement of beetles from the refuge to the transgenic field. (3) CPB flight take-off frequency will be higher on potato plants at the bloom than at the vegetative stage; planting of the non transgenic potato crop in the refuge earlier than the Bt transgenic potato crop in the main field could therefore be considered to increase movement of the Colorado potato beetles from the refuge to the transgenic field. (4) The aggregated distribution of CPB populations in the potato crop is caused by the presence of mating pairs; strategies changing the distribution of males and females in the refuge field could therefore be considered to increase dispersal from the refuge to the main crop field. / Population monitoring using plant counts, flight interception traps, flight landing traps and pitfall traps established that a transient population of adult CPB is present in the transgenic potato fields throughout the crop season and that the abundance of the beetle is higher than that required by the high dose/refuge strategy models. Furthermore results showed that the beetles invading the transgenic field population originate as much from the surrounding fields of conventional cultivars as from the adjacent refuges. It would therefore be possible to relax existing requirements for the refuge to be located immediately adjacent to the transgenic crop. / Like most insects, the adult CPB populations are aggregated and can be fitted to a negative binomial distribution over the crop season. This study revealed that the distribution results from the presence of mating pairs for the overwintered population and from the clumped pupation for the non breeding summer population. The activity of the males in search of females is at least partly responsible for the higher dispersal activity observed with the overwintered than with the summer populations. The comparatively low level of dispersal activity with the summer population could affect the efficacy of the high/dose refuge strategy during the later part of the crop season. / Results of flight chamber tests demonstrated that plant phenology and intraspecific competition have a positive effect on flight take-off frequency. These findings suggest that summer adult dispersal between the refuges and the transgenic crops could be stimulated by manipulating planting dates and the abundance of the different CPB life stages on the plants. / Together, the results of the thesis provide support for some of the premises of the high dose/refuge strategy and offer new information on the CPB dispersal that could be used to further improve its efficacy. Although the transgenic potato (NewLeaf) is not commercially available at this time, the threat of CPB resistance to new products or resistant cultivars under development makes it important to continue the research required by CPB resistance management plans.

No description available.