Potato (Solanum tuberosum) is regarded as one of the single most important vegetable crops in South Africa, with an average annual production of 2 million metric tons. The potato industry contributes to an average of $ 0.37b worth of potatoes annually. Over the years, potato production in South Africa has been affected by, among other factors, diseases and plant parasitic nematodes particularly root-knot nematodes (Meloidogyne spp.). In infected potato fields, root-knot nematodes cause great damage to the crop leading to substantial losses in yield and compromised produce quality. The direct and indirect damage caused by Meloidogyne species results in revenue loss due to a high number of table and processing potatoes rejected in markets both locally and internationally. The presence of resistance breaking Meloidogyne populations, the withdrawal of methyl bromide and lack of commercially grown resistant cultivars suggests that growers are likely to experience more losses in the future. Furthermore, distribution of seed tubers harbouring root-knot nematodes, which may also be asymptomatic, inadvertently facilitates transmission of these parasites to new areas thus perpetuating the problem. Therefore, for the potato industry to adequately address the threat of root-knot nematodes, accurate identification and quantification of root knot nematode levels in the field as well as in seed tubers is of importance. Currently most methods of identifying Meloidogyne species largely rely on the use of morphological traits. However, it can be a challenge to accurately differentiate between closely related species using morphology and other classical methods. To resolve this, recent trends globally have focused on the development of DNA-based diagnostics to rapidly and accurately identify different Meloidogyne species. This study therefore sought to; (a) develop a PCR-based diagnostic tool for accurate detection and identification of various Meloidogyne species parasitizing potatoes in South Africa; (b) use this tool to map their distribution and; (c)develop real-time PCR (qPCR) techniques for accurate quantification and characterization of tropical Meloidogyne species from infected potato tubers. In this study, of the 78 composite potato tuber samples collected from various potato growing regions across seven provinces, 24% were found infected with M. javanica, 23% with M. incognita, 17% with M. arenaria, 14% with M. enterolobii, 3% M. chitwoodi, 1% M. hapla and 1% as M. artiellia. The identity of the remaining 17% could not be established. The three tropical species; M. javanica, M. incognita and M. arenaria were identified as the dominant species, occurring almost in every region sampled. Meloidogyne hapla and M. enterolobii occurred in Mpumalanga and KwaZulu–Natal respectively while M. chitwoodi was isolated from two growers located within the Free State. In the study the use of HRMC and real-time PCR was also developed for identification and quantification of tropical Meloidogyne species infesting potato tubers. Using these two techniques, we were able to show that Meloidogyne arenaria populations produced specific melting peaks (79.3183± 0.0295°C, P < 0.05) thus distinguishing themselves from M. incognita (79.5025± 0.0224°C, P < 0.05) and M. javanica (79.96 ± 0.0459°C, P < 0.05). Real-time PCR was also able to detect 1.53/100th of a nematode using second stage juveniles. / Dissertation (MSc)--University of Pretoria, 2012. / Microbiology and Plant Pathology / unrestricted

Orientador: João Domingos Rodrigues / Resumo: Dois experimentos foram conduzidos em 2018 e 2019 para avaliar os efeitos de diferentes fungicidas de dois importantes grupos: estrobilurinas e carboxamidas, sobre a eficácia fotossintética e produtividade de plantas de tomate, inoculadas com Alternaria sp., em ambiente protegido na Fazenda Ituaú, Salto - SP, Brasil. O delineamento experimental aplicado em ambos os experimentos foi de blocos ao acaso, com oito tratamentos e cinco repetições: testemunha inoculada (água + inóculo), testemunha absoluta (água), azoxystrobina (50,0 g ha-1), boscalida (50,0 g ha-1), azoxystrobina (50,0 g ha-1) + boscalida (50,0 g ha-1), mandestrobina (194 g ha-1), impirfluxam (30 g ha-1), mandestrobina (194 g ha-1) + impirfluxam (30 g ha-1), aplicados em intervalos de 15 dias, totalizando 7 a 8 aplicações no ciclo da cultura. O efeito no desenvolvimento das plantas foi medido pela altura das plantas e produção dos cinco primeiros cachos. A eficiência fotossintética foi calculada pela medição das trocas gasosas, fluorescência da clorofila a, teores de pigmentos fotossintéticos, e produtividade. Os fungicidas apresentaram algumas melhorias nos parâmetros analisados, como melhorando os componentes da eficiência fotossintética e da produção de frutos comerciais, porém sem apresentar diferenças significativas em relação às testemunhas. Os resultados demonstram eficácia no controle de Alternaria sp. aplicando os fungicidas em tomateiro, e também apontam para efeitos adicionais em componentes da eficiênci... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Two experiments were carried out in 2018 and 2019 to evaluate the effects of the application of different fungicides from two important groups: strobilurins and carboxamides, on the photosynthetic efficiency and yield of tomato plants, inoculated with Alternaria sp., grown in greenhouse at Fazenda Ituaú, Salto - SP, Brazil. The experimental design applied in both trials was randomized blocks with eight treatments and five replicates: inoculated control (water + inoculum), absolute control (water), azoxystrobin (50,0 g ha-1), boscalid (50,0 g ha-1), azoxystrobin (50,0 g ha-1) + boscalid (50,0 g ha-1), mandestrobin (194 g ha-1), inpyrfluxam (30 g ha-1), mandestrobin (194 g ha-1) + inpyrfluxam (30 g ha-1), applied at intervals of 15 days, totalizing 7 or 8 applications in the crop cycle. The effect on plant development was measured by plant height and the production of the five initial bunches. The photosynthetic efficiency was calculated from measurements of gas exchange, chlorophyll a fluorescence, content of photosynthetic pigments, and tomato productivity were evaluated. All fungicides positively affected some evaluated parameters, improving photosynthetic efficiency components and commercial fruit production, however without significant difference to the controls. The results demonstrate the efficacy of the use of these fungicides in controlling Alternaria sp. in tomato and also appoint to some additional effects on photosynthesis efficiency components and antioxidant enzym... (Complete abstract click electronic access below) / Doutor

Orientador: Leonardo Bianco / Resumo: Sendo a beldroega uma planta infestante da cultura do tomate e uma hospedeira natural de nematoides do gênero Meloidogyne, levanta-se a hipótese de que a ocorrência dessa planta daninha em áreas de plantios de tomate possa interferir no crescimento e produtividade do tomateiro, dependendo do período de convivência, e que existe uma relação da sua ocorrência com a infestação de M. incognita no tomateiro, e que é possível controlá-la com seletividade ao tomateiro. Dessa forma, com este estudo, objetivou-se: a) avaliar se há interferência da beldroega no crescimento e produtividade do tomateiro; b) determinar o período de convivência da planta daninha com a cultura que não afete o crescimento e a produtividade do tomateiro; c) avaliar se a beldroega infestando a cultura do tomate atua como hospedeira de Meloidogyne javanica e com isso reduz a sua infestação no tomateiro; d) estudar a seletividade de herbicidas ao tomateiro visando o controle da beldroega em pré e pós-emergência e) verificar se a velocidade de controle do metribuzim elimina de imediato a interferência dessa planta daninha no tomateiro. Para tanto, esse estudo constou de três experimentos. No primeiro, os tratamentos experimentais consistiram de três períodos de convivência do tomateiro „Dylla‟ com a beldroega (14, 21 e 60 dias após o transplantio), com um tratamento para tomate sem convivência com a planta daninha e outro tratamento para a beldroega sem convivência com a cultura, todos com ou sem a inoculação de ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Purslane is an infesting plant in tomato culture and a natural host of nematodes of the genus Meloidogyne, the hypothesis arises that the occurrence of this weed in tomato plantation areas may interfere in tomato growth and productivity depending on the period coexistence between them. Also that is a relationship between it ́s occurrence and the infestation of M . incognita in tomato and that it is possible to control it with tomato selective products. This study aimed to: a) assess whether purslane interferes with tomato growth and productivity; b) determine the period of coexistence of the weed with the crop that does not affect the growth and productivity of the tomato; c) to evaluate whether purslane infesting the tomato crop acts as host of Meloidogyne javanica and thereby reduces its infestation in tomato; d) to study the selectivity of herbicides for tomato aiming at the control of purslane in pre and postemergence e) to verify if the speed of control of the metribuzim immediately eliminates the interference of this weed in the tomato. This study consisted of three experiments. In the first, the experimental treatments consisted in three coexistence periods of tomato 'Dylla' with purslane (14, 21 and 60 days after transplanting), with a treatment for tomatoes without living with weeds and another treatment for purslane without coexistence with culture, all with or without Meloidogyne javanica inoculation. The treatments were arranged in a randomized block design, in 6 ... (Complete abstract click electronic access below) / Mestre

La producción de alcohol se encuentra relacionada directamente con la producción de Biocombustibles, debido al desplazamiento de importantes volúmenes de alcohol para su conversión en etanol o alcohol anhídrido; usado como complemento o sustituto de la gasolina. Esta situación ha generado que la caña de azúcar, principal materia prima para la elaboración de alcohol se convierta en un “bien escaso” y sea necesario buscar otras potenciales fuentes para su producción. En el Perú, existe también el potencial para producir el mencionado producto, usando los tubérculos como base, especialmente la papa; a través de la conversión de almidones en azúcares y su posterior fermentación y destilación en alcohol etílico. La presente investigación tiene por finalidad demostrar la viabilidad de la instalación de una planta de producción de alcohol de papa en la provincia de Chota, departamento de Cajamarca, con la intención de atender al mercado interno, con un producto de alta calidad, orientado al uso industrial, farmacéutico y cosmético. La metodología desarrollada comprende la determinación de viabilidad de Mercado, Organizacional, Técnico Operativa, Económica financiera y ambiental. Finalmente se concluye que es viable la instalación de una planta de producción de alcohol en la provincia de Chota.

Phytophthora erythroseptica Pethyb. is a devastating fungal pathogen of potato (Solanum tuberosum). The pathogen causes a disease known as pink rot. Pink rot results in necrosis and decay of tubers; and is responsible for major losses pre and post harvest. Attributes of the disease are progressing toward epidemic proportions. To help prevent such dramatic outbreaks of the disease, understanding the factors associated with incidence will provide opportunities to control the pathogen. A link between pH and disease severity has been observed. We studied the effects of pH and Ca to determine their influence on disease development. Low pH and Ca deficiencies are often inter-related factors that can be causal of one another. The pH effect could be due to either H+ or Ca ions. To separate their interactive effects we tested each variable individually using hydroponics and nutrient solutions. We assessed disease severity by assigning an Infection Coefficient (IC) to each root and stolon samples. The IC values were determined by quantifying DNA and then creating a ratio of host to pathogen DNA within root tissue. The DNA was measured by quantitative RT PCR. Statistical analysis showed significance in greater pathogen presence at more acidic pH and lower levels of available Ca. Significant reductions in IC values were observed when pH was elevated above pH 7. There was a notable increase in colonizing pathogen DNA at pH 5. Ca was significant, and as levels of soluble Ca increased the degree of disease severity became smaller. The effect of Ca was found not to be dependent upon pH. These results suggest immediate and cost effective applied management strategies to reduce incidence and disease severity outbreaks. Amending the soil with lime to increase soil pH and Ca content is one such potential method.

Pathogen derived resistance (PDR) is a recent breakthrough where plant hosts can be made to be resistant to viral infections through transformation with conserved viral genes. Given the severity of Tospovirus diseases worldwide (particularly in tomato), PDR has the potential to garner large yield returns where pathogen populations have overcome the established resistance. Tomato breeding lines FLA7804, FLA8044, and the research line MP1 were used in transformation experiments with potions of the Tomato spotted wilt virus (TSWV) N-gene, and two other chimerical viral nucleocapsid gene constructs from, Impatiens necrotic spot virus (INSV), and Groundnut ringspot virus (GRSV). We conducted 19 independent transformations consisting of 300 to 700 14-day old whole cotyledons each for a total number of approximately 9,000 potentially transformed explants. Of those, approximately 6,300 explants failed to produce regenerants, 2,419 explants underwent abnormal development on elongation media, 187 failed to root, and 215 plants to be characterized genetically. Of the 215 plants, 9 were from FLA 7804, 96 from FLA 8044, and 110 from MP1. Both PCR and Southern blot hybridization analysis later confirmed that none of the 215 plants were transgenic. Opposite to tomato, we were able to transform Arabidopsis thaliana ecotype wassilewskija (Ws) via floral dip with the above listed constructs demonstrating that constructs were not deleterious within a plant once fully introgressed. Sixteen independent transformants in the T0 generation resulted from 19,000 germinated seed from three dipped plants resulting in a total transformation rate of 0.08%. Of the 1,000 T1 seed germinated on kanamycin media from each of the 16 putative Arabidopsis plants transformed with the construct containing elements of the N-gene from all three of the aforementioned tospoviruses, four populations exhibited simple Mendelian inheritance of the transgene. DNA walking analysis yielded amplification of the unknown region outside the nptII region of the insert for three of the four remaining transformants, which was subsequently sequenced and mapped to chromosomes 1, 3, and 4. There were 25 T1 individuals selected from each population and transferred to soil for DNA extraction and zygosity determination. Homozygous T2 seed was collected for future resistance studies.

The domesticated tomato Solanum lycopersicum (L.), formerly known as Lycopersicon esculentum is a genetically well-studied crop species with high-density linkage and molecular maps based on crosses done between cultivated tomato and its distant related wild species. Wild tomato species harbor a wealth of resistance to many pathogens that have been introgressed into domesticated tomato for genetic control of diseases and pests and for improvement of many agronomic traits. The wild tomato S. peruvianum (L.) is the source of the Sw-5 gene, characterized and mapped to chromosome 9 of the tomato genome and introgressed into elite tomato germplasm, providing resistance to the tospovirus Tomato spotted wilt virus (TSWV). TSWV has been reported to be a major problem for tomato growers in many parts of the world, which in some cases, has resulted in tomato fields having been abandoned for some time. Additionally, there are reports that new races of TSWV have evolved that overcome Sw-5. TSWV replicates in both, plant cells and in the alimentary canal cells of thrips and then transmitted to plants by this insect acting as a vector. Both, TSWV and thrips have co-evolved to infest and infect more than 1090 plants species in over 100 families, thrips becoming resistant to pesticides and easily escaping by hiding deep in plant parts. World trade has disseminated thrips all over the world and environmental pressures have forced TSWV to recombine its RNA to overcome new resistance.

Diploid potato populations between a primitive cultivated species, <I>Solanum phureja</I>, and a weedy species, <I>S. chacoense</I>, were used to examine the segregation of microsatellite markers and three traits in backcrosses. Two of the traits, anther culture competence and 2<I>n</I> pollen production, originated from <I>S. phureja</I> whereas the third, leptine production (a specific glycoalkaloid known to convey resistance to the Colorado potato beetle) originated from <I>S. chacoense</I>. Using CP2, a self-incompatible F₁ hybrid originating from a cross between <I>S. chacoense</I> clone 80-1 and <I>S. phureja</I> clone 1-3, three populations were developed: 1-3 x CP2 (PBCp), CP2 x 1-3 (PBCc), and CP2 x 80-1 (CBC). For the microsatellite study, four simple sequence repeat (SSR) primer pairs that amplified fragments within potato sequences found in the GenBank were used to look at segregation ratios in our backcross populations and to eliminate possible spurious genotypes bearing non-parental alleles in these populations. Seventeen spurious genotypes were discarded from PBCp; none was found in PBCc or CBC. Two SSR loci showed skewed segregation in PBCp (favoring transmissnion of the allele originally found in 80-1), PBCc showed normal segregation at all loci, and CBC showed distorted segregation at one locus (revealing a deficiency of homozygotes). In the study of anther culture, three components of ACR were investigated in a preliminary study: 1) embryos produced per anther (EPA), 2) embryo regeneration rate and 3) percentage of monoploids (2<I>n</I>=1<I>x</I>=12) among regenerants. CP2 was intermediate, 80-1 was low, and 1-3 was high for ACR. Only EPA was selected for further characterization in our populations. PBCp (78 genotypes) and CBC (57 genotypes), were characterized for anther culture response ACR/EPA in a series of studies. Nine high and ten low selections were identified in CBC, and ten high and ten low selections were identified in PBCp. EPA selections were used for bulk segregant analysis (BSA) using 214 RAPD primers. Two bands, one amplified by OPQ-10 and another by OPZ-4 were linked in coupling and in repulsion, respectively, to ACR in PBCp. One band amplified by OPW-14 primer was linked in coupling to ACR in CBC. One-way ANOVAs for data from remaining genotypes of the populations verified linkage of the markers to ACR/EPA. For 2n pollen production, a total of 77 PBCp genotypes was characterized; 80-1 produces low % 2<I>n</I> pollen, and 1-3 produces high % 2<I>n</I> pollen. Pollen samples were stained with propidium iodide and examined by flow cytometry. The frequency of 2n pollen varied continuously from 1.7 % to 40.6 % among the 41 genotypes that flowered sufficiently to allow three separate pollen collections. Variation due to the environment was observed where the frequency of 2n pollen appeared greater over a range of genotypes on single collection days. BSA could not be used due to limited population size and a low number of selections at the extremes of the distribution of phenotypes. The continuous variation for 2<I>n</I> pollen production suggests multigenic control of the trait. In the study of leptine content in reciprocal backcross populations, 87 genotypes within PBCp, and 42 genotypes within PBCc were characterized using gas chromatography of leaf samples. CP2 was intermediate, 1-3 had zero, and 80-1 was high for leptine content in the foliage. Leptines were present in low levels in 43 of 87 genotypes in PBCp, indicating simple genetic control. In PBCc, only 7 of 42 genotypes expressed leptines, generally at a higher level than in PBCp, indicating cytoplasmic inheritance. Ten high and ten nil selections within PBCp, and seven high and eight nil selections within PBCc were used for BSA using 214 RAPD primers. Three primers OPQ-2, OPT-16 and OPT-20 amplified bands segregating with high bulks in both populations. These markers were linked in coupling to leptine content in PBCp. Linkage was verified by ANOVAs for leptine content in the entire population. / Ph. D.

Genetic improvement of the potato through classical breeding has been limited by its tetraploid nature, the narrow genetic variability within cultivars, and interploidy barriers between tetraploid cultivars and diploid germplasm. Breeding at reduced ploidy levels has been proposed as a solution to these problems. Because of sterilities, somatic hybridization via protoplast fusion has been considered an alternative to sexual polyploidization for resynthesizing superior diploids from selected monoploids, and tetraploids from selected diploids and dihaploids. Successful application of somatic hybridization largely depends upon protoplast culturability and regenerability of a plant. The ability of callus formation and plant regeneration from protoplasts varies among plants. To understand the genetic basis for this variation, the mode of inheritance for protoplast culturability, defined as the ability to develop calli from cultured protoplasts, was studied in the diploid potato species, Solanum phureja. Based upon data from F₂ as well as from F₁ and backcross progenies, it was found that protoplast culturability in this potato species was controlled by two unlinked loci with dominant effect. In addition, there was quantitative variation for protoplast plating efficiency among culturable genotypes. Male sterility in cultivars of Solanum tuberosum ssp. tuberosum results from nuclear-cytoplasmic interactions. 'Donor-recipient' protoplast fusion and regeneration were conducted between a sterile S. tuberosum ssp. tuberosum cultivar, Russet Burbank, and fertile selections of S. tuberosum ssp. andigena which have a non-sensitive cytoplasm and were used as the cytoplasmic donor. Sixteen regenerated plants possessed nuclear background and chloroplast DNA of Russet Burbank. However, two of these regenerants had improved pollen stainability. The possible causes for the improvement of pollen stainability are discussed. In the last chapter, allelic polymorphism in a monoploid population derived from anther culture of a clone of S. phureja was assessed by isozyme electrophoresis. Fourteen monoploids and their anther donor were examined for six enzymes. No allozyme variation was detected in these plants. However, genetic variability among these monoploids was manifested by variations in some growth characters and general morphology. The limitation of enzymatic markers in detecting allelic polymorphism in these monoploids is discussed. / Ph. D.

The variation for embryo production in anther culture of Solanum phureja was examined as a function of maximum greenhouse temperature prior to bud harvest and innate responsiveness among anthers within a bud. S. phureja clones PP5, AD2-4, A3P2-6 and AD3-4 were grown in a greenhouse under a 16 h photoperiod. The temperature was monitored continuously using a thermograph. Buds were collected from PPS and AD2-4 and the anthers were cultured in two groups of five flasks. In the first group, each flask contained the 30 anthers from 6 buds; the second group, each flask contained 1 anther from each of 30 buds -- a total of 30 anthers per flask. Significantly smaller coefficients of variation were observed for the second group, suggesting that the variation for embryogenic capacity among buds was greater than that among anthers within a bud. Variation in embryo yield as a function of greenhouse temperature for clones A3P2-6 and AD3-4 was examined by stepwise regression analysis. Embryogenic capacity of clone A3P2-6 was adversely affected by high temperatures (31-37°C) that occurred 2 and 7 days before bud harvest. However similarly high temperatures appeared to enhance the androgenic response of clone AD3-4. Regeneration rate of anther-derived embryos over three subcultures to fresh regeneration medium was examined as a function of anther donor or clone, cold pretreatment of embryos, and morphological classification of embryos. Only clonal origin significantly affected regeneration. Regeneration rate declined on each serial subculture. The frequency of regenerable embryos varied from 12.5% for clone BARD 1-3 to 46.0% for clone A3P2-6. Flow cytometric analyses were performed on several anther-derived monoploids of S. phureja to examine the frequency of nuclei at the 1x, 2x, and 4x levels within and among clones. Significant variation was found among duplicate cultures of the individual clones, but this variation was small enough to allow the detection of significant differences among the clones. Monoploid cell frequency ranged from 22.3% to 35.7%. Diploid cell frequency ranged from 48.6% to 59.9%. Tetraploid cell frequency ranged from 11.9% to 25.3%. Several families of anther-derived monoploid clones of S. phureja were analyzed for differences among clones within a family and among families. Significant differences were found in both categories. Finally, unstained protoplasts of monoploid S. phureja clone AM3 were sorted based on forward angle light scatter (FALS) and autofluorescence. Fractions selected for low FALS and weak autofluorescence appeared to be selectively enriched for monoploid protoplasts. / Master of Science