Colletotrichum acutatum and C. gloeosporioides are fungal plant pathogens that have a global distribution, extensive host range, and convoluted taxonomy. Both species can cause grape ripe rot and are considered endemic to Virginia US. In 2012, C. acutatum and C. gloeosporioides were reclassified into species complexes that consist of 31 and 22 accepted species, respectively. The objectives of this study were to: 1) survey Virginia vineyards for grape ripe rot, and morphologically and phylogenetically identify isolates to the species within the complexes, 2) conduct an in vitro fungicide assay to screen fifteen commercial fungicides and combinations of two fungicides for efficacy to control isolates from seven Colletotrichum species from Virginia vineyards, 3) sequence gene fragments from three subunits of the SDH enzyme in the fungicide-screened isolates to observe potential resistance mutations, 4) investigate the susceptibility of three grapevine tissues to Colletotrichum species, 5) observe potential infection structures before and after the application of fungicides, 6) evaluate the efficacy of commercial fungicide controls of grape ripe rot in the field, and determine the most advantageous timing of applications. In my studies, I identified six Colletotrichum species: C. aenigma, C. conoides, C. fioriniae, C. gloeosporioides, C. kahawae, and C. nymphaeae. I also found two additional groups; an isolate similar to C. limetticola and C. melonis and a group of isolates that are similar to C. alienum, C. fructicola, and C. nupharicola. I also identified captan, and mancozeb as two potential active ingredients for control of grape ripe rot isolates from Virginia via the in vitro fungicide assay. Additionally, I found that combinations of two active ingredients could increase the efficacy of benzovindiflupyr, copper, and polyoxin-D. C. fioriniae germination and production of melanized appressoria was documented on leaves. I observed appressorium formation with isolates of two C. fructicola-like genotypes and C. nymphaeae, as well as secondary conidiation with isolates of C. aenigma, C. fructicola-like genotype 3, and C. nymphaeae on blooms. And finally, benzovindiflupyr, cyprodinil + fludioxonil pre-mix, and potassium phosphite + tebuconazole were identified as candidates for chemical control for grape ripe rot in the field. / PHD / Colletotrichum acutatum and C. gloeosporioides are two fungal plant pathogens that are found on a wide range of crops around the globe. Both fungal species cause the disease grape ripe rot and have been found in Virginia (VA) USA since the late 1800s. Originally, grape ripe rot was considered a minor disease in VA; however, based on communications with local VA vineyard managers, grape ripe rot was found to cause up to 30% direct crop loss. Further indirect economic loss occurs during wine production due to the production of unpalatable, tobacco-like, off flavors from the infected grapes. Sensory studies found this wine flavor change occurred with as little as 3% of the total crushed grapes being infected. Grape ripe rot appears as a sunburn-like, tan injury on the surface of white-fruited grape berries. As the disease progresses, the dark injury expands across the surface of the berry and rings of salmon-colored spore masses form. On red-fruited grapes, the formation of spore masses is usually the first observable sign. Over time, the infected berries will shrivel down to a soft, pustule-covered raisin. Both C. acutatum and C. gloeosporioides cause the same grape ripe rot symptoms on fruit and overlap in fungal appearance. In addition, investigations of these pathogens using molecular techniques have revealed that each consists of a number of genetically distinct groups that are difficult to distinguish by appearance. Therefore, in 2012, C. acutatum and C. gloeosporioides were reclassified into 31 and 22 newly accepted species, respectively, using molecular techniques. The objectives of this study were to: 1) survey VA vineyards for grape ripe rot, and visually and molecularly identify isolates to the species within the new complexes, 2) conduct a laboratory fungicide assay to screen fifteen commercial fungicides and combinations of two fungicides for control of isolates from VA vineyards, 3) sequence gene fragments from three subunits of the SDH enzyme in the fungicide-screened isolates to observe potential resistance mutations, 4) investigate the susceptibility of three grapevine tissues to Colletotrichum species, 5) observe potential infection structures before and after the application of fungicides, 6) evaluate the efficacy of commercial fungicide controls of grape ripe rot in the field, and determine the most advantageous timing of applications. In my studies, I identified six Colletotrichum species: C. aenigma, C. conoides, C. fioriniae, C. gloeosporioides¸ C. kahawae, and C. nymphaeae. I also found two additional groups; an isolate similar to C. limetticola and C. melonis and a group of isolates that are similar to C. alienum, C. fructicola, and C. nupharicola. Our lab also identified four active ingredients as potential controls of grape ripe rot in the laboratory fungicide assay; captan, mancozeb, tetraconazole and thiophanate-methyl. Additionally, combinations of two compounds can increase the effectiveness of benzovindiflupyr, copper, and polyoxin-D. of C. fructicola-like isolates, and C. nymphaeae formed infection structures on blooms. of C. aenigma, C. fructicola-like genotype 3, and C. nymphaeae formed spores on blooms without producing symptoms. C. fioriniae spores germinated and produced infection structures on leaves without producing symptoms.

Stink bugs, including native brown stink bug (Euschistus servus) and invasive brown marmorated stink bug (Halyomorpha halys), cause damage to a variety of crops including field corn (Zea mays). Frequency and size of stink bug infestations have increased in corn fields in the Mid-Atlantic U.S., and there are growing concerns that these infestations may contribute to reductions in grain quality including increased mycotoxin concentrations. Prior research on native and invasive stink bugs has focused on understanding their biology, the damage they cause, and elucidating effective and economic management strategies. However, few studies examined the potential for stink bugs to facilitate fungal infection and mycotoxin contamination of corn grain. Thus, the objectives of this research were to: 1) assess the relationship between invasive brown marmorated stink bug (H. halys) feeding injuries and fumonisin contamination of field corn in the Mid-Atlantic U.S., 2) determine if stink bugs are a vector for mycotoxigenic Fusarium spp. in corn, and 3) evaluate the efficacy of pesticides for mitigating stink bug feeding injury and associated mycotoxin contamination in field corn. A correlation between H. halys feeding injury and fumonisin concentrations was identified, and the ability of H. halys to increase F. verticillioides infection and fumonisin concentrations in corn was demonstrated in field experiments. Fusarium species including fumonisin-producing F. verticillioides and F. proliferatum were isolated from field-collected stink bugs, and in laboratory experiments, E. servus was able to transmit F. verticillioides to non-infected corn ears after feeding on F. verticillioides-infected corn. In field studies, both fungicide and insecticide reduced stink bug-associated mycotoxin concentrations in corn, but levels of control were inconsistent. Thus, additional tactics that target both the stink bug and Fusarium should be implemented to mitigate risks of mycotoxin contamination in corn. / Doctor of Philosophy / Native and invasive stink bugs can severely damage crops including field corn. Frequency and size of stink bug infestations in Mid-Atlantic U.S. corn fields have increased, and there is growing concern that this may contribute to reductions in grain quality. Insect feeding injury is a risk factor for fungal infection and mycotoxin contamination in corn. Mycotoxins are toxic chemicals produced by certain fungi that have detrimental health effects on animals including livestock and humans. The relationship between stink bug feeding injuries and mycotoxin contamination in corn grain is not well understood, and management strategies to minimize the risk of mycotoxin contamination in corn need to be identified. The main goal of this research was to characterize interactions between stink bugs and mycotoxin-producing fungi and identify tactics for controlling both the insect pest and pathogen. Specific objectives were to: 1) assess the relationship between invasive brown marmorated stink bug (H. halys) feeding injuries and fumonisin contamination of field corn in the Mid-Atlantic U.S., 2) determine if stink bugs are a vector for mycotoxin-producing Fusarium spp. in corn, and 3) evaluate the efficacy of pesticides for mitigating stink bug feeding injury and associated mycotoxin contamination in field corn. Results from this work indicated that stink bugs have the ability to cause feeding injuries which facilitate invasion of mycotoxin-producing Fusarium species, leading to increases in mycotoxin concentrations in corn grain. Studies also demonstrated that stink bugs can vector Fusarium species during feeding and increase Fusarium infection of corn resulting in subsequent mycotoxin contamination. Field studies indicated that pesticide applications targeting both the stink bugs and mycotoxigenic fungi may be needed to minimize risk of mycotoxin contamination in corn. However, under low pest pressure, application of pesticides is unlikely to be profitable.

The metabolism of white-rot fungi has many proposed biotechnological applications. Their unique capability to depolymerize and catabolize lignin, the most recalcitrant component of lignocellulosic biomass, could be instrumental to the sustainable production of fuels, chemical, and materials from waste biomass feedstocks. The non-specific, oxidative nature of this lignin-degrading metabolism of white-rot fungi renders them capable of degrading a wide range of complex refractory organic compounds beyond lignin, including emerging micropollutants such as pharmaceuticals and pesticides which current wastewater treatment processes were not designed to remove. However, harnessing these metabolic capabilities into engineered bioprocesses has proven to be challenging. Common bioreactor design strategies were developed for traditionally-used unicellular bacteria and yeasts and are not necessarily appropriate for the more complex, filamentous white-rot fungi. Due to a lack of specific engineering strategies and other knowledge gaps, the realization of white-rot fungal bioprocesses has been hampered by low process efficiencies and operational challenges. This dissertation aims to expand the engineering toolbox for harnessing the metabolism of white-rot fungi in bioprocesses. Specifically, it proposes the addition of Fenton chemistry as an avenue to unlock the biotechnological potential of white-rot fungi. The production of hydroxyl radicals through the Fenton reaction is generally understood to be part of the lignin-degrading machinery of white-rot fungi and the addition of Fenton chemistry has been shown to synergistically enhance lignin degradation by white-rot fungi. Overall, the research presented here aims to demonstrate that incorporating Fenton chemistry into white-rot fungal bioprocesses not only synergistically increases lignin degradation efficiency, but also offers a potential solution for the operational challenges that have prevented the implementation of white-rot fungal bioprocesses. This dissertation was guided by five objectives aimed at illustrating the utility of coupling Fenton chemistry and white-rot fungi in engineered bioprocesses. The first objective was to demonstrate, optimize, and uncover the underlying mechanisms driving the synergistic degradation of lignin by white-rot fungi and Fenton chemistry. Through this assessment, it was found that lignin degradation increased synergistically from 58.8% to 80.2% in the presence of Fenton chemistry at the optimum concentration. This work also showed that Fe(II)/Fe(III) cycling and the induction of auxiliary ligninolytic pathways mediate this synergistic interaction. The second objective was to elucidate how Fenton chemistry influences the regulating mechanisms of ligninolytic activity in white-rot fungi, specifically C:N ratio. This showed that C:N ratio significantly influences lignin degradation in the absence of Fenton, but that this effect is blunted in the presence of Fenton. The third objective was to investigate how Fenton chemistry modulates the relationship between the concentration of fungal biomass and the extent of lignin. In the absence of Fenton, fungal biomass concentration was strongly correlated to the extent of lignin degradation. While this was also the case in the presence of Fenton chemistry at very low fungal biomass concentrations, this relationship became uncoupled at sufficiently high fungal biomass concentrations. The fourth objective was to evaluate Fenton chemistry as a selective disinfectant to allow for the persistence or enrichment of white-rot fungi in non-sterile settings. The model competitor E. coli became completely inactivated within hours at the optimal concentration of Fenton reagents, whereas the white-rot fungus P. chrysosporium survived and grew. Lastly, the fifth objective was to demonstrate the long-term performance of a continuously-operated bioreactor which integrated Fenton chemistry and white-rot fungal metabolism. A rotating biological contactor (RBC) combined with a rotating cathode electro-Fenton was constructed and a kinetic model based on batch tests was successfully developed and validated. The reactors were operated for over 100 days and reached stable lignin degradation performance at ~55%. Analysis of the microbial ecology of these reactors showed the persistence of the inoculated P. chrysosporium within the biofilms, as well as the enrichment for other lignin-degrading fungi and bacteria with aromatic catabolism and iron-reduction capabilities. Overall, this research provides insight into the potential and practical implications of integrating Fenton chemistry with white-rot fungi in bioprocesses. The lignin-degrading metabolism of white-rot fungi has long been of interest for biotechnological purposes, but attempts to operationalize them have thus far been unsuccessful at scale. In order to consider scaling white-rot fungi to full-scale operations such as wastewater treatment plants, a better understanding and tighter controls on the growth, ligninolytic activity, and ecological interactions of white-rot fungi are needed. This work proposes Fenton chemistry as a synergetic actor, selective promoter and regulator of white-rot fungal biomass and their production of lignin degrading enzymes.

A new selective medium (TB-CEN) was developed for isolating Thielaviopsis basicola, cause of black root rot of tobacco, from soil. TB-CEN medium contains etridiazol and nystatin to inhibit the growth of undesired fungi, and unautoclaved extract from carrot to selectively enhance for the growth of T. basicola. Inoculum and/or population densities of T. basicola in five burley tobacco fields were 74-166 propagules per g of soil, and 0-12 propagules per g of soil in three other burley fields. Inoculum and/or population densities of T. basicola in 12 flue-cured and 2 sun-cured tobacco fields were 0-26 propagules per g of soil, and 101 and 402 propagules per g of soil in two other flue-cured fields. Environmental factors apparently had a strong effect on black root rot development, since root rot and plant stunting were severe in two burley fields that had 148 and 158 propagules per g of soil, but were not severe in the two flue-cured fields that had 101 and 402 propagules per g of soil. All of the cultivars planted in the four fields were susceptible. Black root rot was the major disease associated with the stunting of tobacco plants in the burley region of Virginia, but not in the flue- and sun-cured regions. No evidence was found to indicate that endomycorrhizae were involved in tobacco stunting in Virginia. T basicola inoculum density-disease severity studies were conducted both in soil-temperature tanks and in the field. Tobacco seedlings were grown in temperature tanks (20-23 C) for 30-31 days in naturally infested field soil (pH 6.5). For all cultivars tested (Burley 21, NC95, and Va Gold), the mean percent of roots that were rotted increased significantly (P=0.001) as inoculum density increased (R² range for regressions=0.93-0.97). Severe levels of root rot occurred at inoculum densities of 50-200 propagules per g of soil. Significant (P=0.01) reductions in plant growth occurred at inoculum densities as low as 5-10 propagules per g of soil. In a study conducted on a commercial burley tobacco (cv. B21-Ky10) field, inoculum densities of 150 and 683 propagules per g of soil were associated with moderate and severe levels of black root rot, respectively. Differences between soil-temperature tank and field studies appeared to be due to variations in environmental- and host-related factors. In another burley field study, the fungicide imazalil, which completely inhibited the growth of T. basicola when amended into agar media at a concentration of 1.0 μg a.i./ml, failed to control black root rot when it was added to transplanting water (50 ml/plant) at concentrations as high as 1,500 μg a.i./ml. / Ph. D. / incomplete_metadata

京都大学 / 新制・課程博士 / 博士(農学) / 甲第25315号 / 農博第2581号 / 新制||農||1103(附属図書館) / 京都大学大学院農学研究科農学専攻 / (主査)教授 白岩 立彦, 教授 那須田 周平, 教授 田中 千尋 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

Vitis vinifera L.(Vv), the European cultivated grapevine is one of the most worldwide important crops but is highly susceptible to the necrotrophic fungus Botrytis cinerea (Bc), the causal agent of bunch rot (BR) disease. In grapevine, as well as in other fruit species, it has been described a primary infection by Bc at full bloom, followed by a quiescence period during the berry development, and the fungal egression after veraison reaching the maximum at harvest. Today, this important disease is mainly controlled by massive use of fungicides, which are applied at different developmental stages that happen to be critical during the grapevine-Bc interaction. During the contact, the fungus must overcome several barriers from the host, which protect it from the pathogen attack and might be also modulated or activated due to the presence of the pathogen itself. The cuticle and the cell wall (CW) represent the first barriers from the plant encountered by the pathogen. To successfully colonize the plant tissue, Bc possesses several virulence factors, and CW modifying enzymes (CWMEs) are part of them. On the other hand, the regulated activity of the CWMEs, expressed both by the host and the pathogen, could alter the plant CW composition and porosity, therefore facilitating, or limiting the penetration of the fungus. Among the CWMEs, Pectin methylesterases (PMEs) regulate the degree of methyl esterification of the pectin homogalacturonan, also modifying the epitopes for the activity of other CWMEs such as polygalacturonases (PGs) and pectate lyases (PLs) by whose action, pectin becomes more susceptible to degradation and the CW more accessible by the pathogen. Previous works both in Arabidopsis thaliana and in crop species identified several PME genes with an altered expression in response to pathogens. A previous report characterizing Atpme17 mutant lines has highlighted a role of AtPME17 in the resistance response to Bc in contrast with the opposite role of AtPME3, suggesting that PME genes could have a completely different action during Bc response depending on the isoform involved. In this context, the main objective of the project was to identify new strategies for Bc BR-control, and specifically i) to identify candidate genes involved in the response to Bc, whose inactivation/overexpression would lead to Bc resistant plants and ii) to set up a molecular method to monitor the Bc load in the field and therefore implement a more sustainable control of the pathogen. To further understand the effects of the Bc primary infection in grapevine flowers at CW level, two contrasting genotypes (Souvignier gris (SG) and Teroldego (TE)) in their resistance to the fungus were considered. An artificial inoculation of different biological replicates, in vase maintained, was performed at full bloom, in controlled conditions, and samples were collected at 24 hours post-inoculation (hpi) with the fungus and post-treatment with the respective control, for the following RNA-seq analysis and biochemical characterization of PME activity and CW composition in the two genotypes before and upon infection. The Bc load was estimated in the flowers using qPCR and as expected, a higher biomass of Bc was found in TE, the susceptible cultivar, than in SG, the resistant one. The analysis of CW composition, PME activity and degree of pectin methyl-esterification, both in treated and control flowers, showed significant differences between the two genotypes, in particular SG showed a significant induction of PME activity with respect to the control, evidence not present in the susceptible genotype. The RNAseq analysis on the same samples showed a total of 4800 genes modulated, out of which 3064 are only modulated in TE, 739 only in SG and a common group of 997 genes. Regardless of the cultivar, upon infection there was a total 2919 genes upregulated vs 1909 genes downregulated. A gene set enrichment analysis (GSEA) indicated several over-represented categories upon infection, including response to pathogens and biosynthesis of secondary metabolites, with a general down-regulation of those genes related to CW organization and pectin modification (CWMEs), mostly in the resistant genotype. Within the down-regulated CWMEs, Pectin methylesterase (PME) genes were found highly represented. Unlike, a larger gene set, in many cases with a higher fold-change of induction, was identified in TE respect to SG. This is the case of genes involved in the defense response and its regulation, and in the modification/reinforcement of the cell wall, therefore attesting for an initial tentative by the susceptible genotype to counteract the pathogen, although at the end without success. This was also the case of the seven VviPME genes previously highlighted by the in-silico co-expression analysis and therefore of VviPME10, the gene with the highest homology to AtPME17. Among the regulators, one WRKY factor (VviWRKY3), known to be related to defense response in grapevine mediated by stilbene synthesis, was also further characterized as putative regulator of VviPME10, whose promoter hosts more than one several predicted binding sites for VviWRKY3. Indeed, luciferase assay results indicate a significant activation of VviPME10 promoter by VviWRKY3 factor. Parallelly, the genome-wide analysis of the last structural annotation of the grapevine genome assembly allowed us to identify 62 VviPME gene members, 15 more than a previous report, and manually curate the gene structure for 39 of them. Then, to corroborate the idea of the role of the CW, and in particular of PME activity, in the grapevine response to the fungus, an in silico co-expression analysis of the 62 VviPME members, considering the publicly available RNA-seq experiments related to grapevine-Bc interactions and the RNAseq experiment conducted in this project, was performed. The analysis highlighted a group of seven genes (VviPME1, VvPME9, VviPME10, VviPME11, VviPME12, VviPME13 and VviPME54) with significant induction upon Bc infection, five of them (VviPME8, VviPME9, VviPME10, VviPME11, and VviPME54) located in the same chromosome (chr06). VviPME10 showed the highest homology and was found to be phylogenetically close to the Arabidopsis thaliana PME17 gene, suggesting being considered as its putative orthologue. Afterward, Therefore, considering the increased VviPME10 expression upon infection, and the reported effect of AtPME17 in A. thaliana, VviPME10 was selected as a potential candidate to study its role in grapevine. In this regard, two strategies were adopted, i. VviPME10 knock-out (KO) with CRISPR/Cas9 and ii. VviPME10 overexpression (OE, under CaMV35S promoter) through embryogenic callus transformation of the grapevine cultivar ‘Sugraone’ mediated by Agrobacterium tumefaciens. More than 100 embryos developed, and around 20 plantlets per transformation were analyzed to check the presence of the transgenic construct. Then, the mutation profile, in the case of KO lines, and the expression analysis of the transgene, in the case of OE lines, were carried out to select the appropriate lines to acclimatize. OE lines were also tested for VviPME10 activity. A total protein extract was obtained from the leaves of the lines, showing a higher protein activity compared to the control, and indicating the functionality of the enzyme. Unfortunately, grapevine OE lines couldn’t be analyzed for their response to Bc, while KO lines showed a significantly larger lesion area when compared to the control at 5 days post fungal inoculation (dpi). However, the effect of VviPME10 overexpression upon Bc infection was evaluated also in Nicotiana benthamiana VviPME10-OE lines, generated in parallel. At 3 dpi a significant reduction was observed in the lesion area compared to the control. These results suggest that pectin modification, mediated by VviPME10, plays an important role in the grapevine response to Bc, in particular it seems to behave more like a resistance gene than a susceptibility one. For this reason, it could be considered as a valuable target to improve resistance to Bc in susceptible grapevine varieties. Finally, a molecular method for Bc detection, based on quantitative RT-PCR assays, was set up and applied to estimate the Bc load in field conditions. Although the method allowed the successful detection of the presence of the fungus in samples at different developmental stages, from two V. vinifera cultivars, in different vineyards, the lack of environmental conditions for the development of the disease might have impaired the correlation between detection and the development of the disease. Nonetheless, the method represents a good alternative for monitoring the Bc load in the field at the early season, to predict the BcBR severity at harvest and eventually apply the disease management protocols based on the real need.

Em cana-de-açúcar, a colonização do caule por fungos oportunistas, como Fusarium verticillioides e Colletotrichum falcatum, está diretamente ligada ao ataque da lagarta Diatraea saccharalis (Lepidoptera: Crambidae). Duas proteínas, SUGARWIN1 e SUGARWIN2 são produzidas em cana-de-açúcar, em resposta ao dano mecânico e ao ataque de D. saccharalis, porém estas proteínas não afetam o inseto, e sim ocasionam alterações fisiológicas e morfológicas em F. verticillioides e C. falcatum, ocasionando a morte destes fungos por apoptose. Dietas artificiais suplementadas com estes fungos oportunistas ocasionaram o ganho de peso da D. saccharalis. Esses dados indicam uma interação mais íntima entre o inseto e estes patógenos de cana, sendo que, neste estudo procuramos identificar relações simbióticas entre os indivíduos, analisando se a forma de transmissão desses fungos é mediado pela D. saccharalis. Os resultados mostraram a presença do F. verticillioides em todas as fases de desenvolvimento da D. saccharalis após contato com o fungo, ou seja, depois de se alimentarem em dieta suplementada por F. verticillioides no 4º instar, permaneceram infectadas pelo fungo ao longo de toda a fase pupal e adulta, em ambos os sexos. Além disso, o F. verticillioides foi transmitido para os descendentes de D. saccharalis, sendo que o fungo foi detectado nos ovos, ou seja, um caso original de transmissão vertical. Por meio de microscopia, também foi possível verificar a alta intensidade de F. verticillioides no interior do intestino de lagartas. Estes dados inferem em uma relação simbiótica entre F. verticillioides e D. saccharalis, onde o simbionte é transferido verticalmente para as gerações subsequentes. As respostas obtidas com o fungo C. falcatum diferiram daquelas obtidas com F. verticillioides, uma vez que não se detectou a presença do fungo a partir da fase pupal. Neste caso, a relação de simbiose entre o fungo e o inseto pode resultar em uma transmissão horizontal. Com este estudo foi possível identificar diferentes formas de transmissão por D. saccharalis para dois fungos envolvidos em podridão de colmo em cana-de-açúcar. Estes dados mudam a forma como é vista a transmissão de F. verticillioides por D. saccharalis em cana-de-açúcar, podendo influenciar a forma de manejo da podridão de Fusarium e da broca nos canaviais. / In sugarcane, stem colonization by opportunistic fungi, such as Fusarium verticillioides and Colletotrichum falcatum, is directly linked to the attack of Diatraea saccharalis (Lepidoptera: Crambidae) caterpillar. Two proteins, SUGARWIN1 and SUGARWIN2 are produced in sugarcane, in response to mechanical damage and attack of D. saccharalis, however these proteins do not affect the insect, but cause physiological and morphological changes in F. verticillioides and C. falcatum, causing the death of these fungi by apoptosis. Artificial diets supplemented with these opportunistic fungi caused the weight gain of D. saccharalis. These data indicate a more intimate interaction between the insect and the sugarcane pathogens. In this study, we sought to identify symbiotic relationship among individuals, analyzing whether the transmission of these fungi is mediated by D. saccharalis. The results showed the presence of F. verticillioides in all stages of D. saccharalis development after contact with the fungus, in the 4th instar. The caterpillars remained infect by the fungus throughout the pupal and adult phase, in both sexes. In addition, F. verticillioides was transmitted to D. saccharalis offspring, being detected in eggs, an original case of vertical transmission. Through the microscopy results, it was also possible to verify the high intensity of F. verticillioides inside the intestines of caterpillar. These data infer in a symbiotic relationship between F. verticillioides and D. saccharalis, where the symbiont is transferred vertically to the offspring. The responses obtained with C. falcatum differed from those obtained with F. verticillioides, since the presence of the fungus was not detected from the pupal phase. In this case, the symbiont relationship between fungus and insect can result in a horizontal transmission. With this study was possible to identify different forms of fungi transmission by D. saccharalis. These data change the way the transmission of F. verticillioides by D. saccharalis in sugarcane is viewed, and may influence the management of Fusarium rot and sugarcane borer attack in sugarcane.

Thesis (M. Sc. (Agriculture, Horticulture)) --University of Limpopo, 2016 / ‘Reed’ avocado is a late season cultivar introduced to South Africa from California. The cultivar has shown good adaptation and produces quality fruit with export potential. Its pre-harvest adaptation and production aspects have been researched and documented. However, the effect of harvest time, post-harvest storage and ripening temperature has not been comprehensively studied on this newly introduced ‘Reed’ avocado cultivar. Therefore, the aim of this work was to investigate the effect of different harvest time, post-harvest storage and ripening temperature on the quality of late season ‘Reed’ avocado fruit. Matured ‘Reed’ avocado fruit were harvested based on moisture content indexing in December (2015) and January (2016). The experiment was carried out in a factorial, arranged in a completely randomised design (CRD) with three replicates. Treatment factors were: 2 x harvest time (mid-and late), 2 x post-harvest storage (2.0 and 5.5°C), 3 x ripening temperature (16, 21 and 25°C) and 5 x ripening day (0, 2, 4, 6 and 8). Fruit were stored at 2.0 and 5.5°C for 28 days, thereafter, ripened at 16, 21 and 25°C until fully ripe. During ripening, fruit were evaluated for weight loss, skin colour, firmness, respiration rate, physiological and pathological disorders. Mid-harvest fruit had higher moisture content when compared with late harvest fruit. However, harvest time, post-harvest storage, ripening temperature and ripening time (days) significantly influenced fruit weight loss, firmness, respiration rate, ripening percentage of ‘Reed’ avocado fruit during ripening. Moreover, fruit firmness decreased faster at higher temperatures (25 and 21°C) with fruit ripening within 4 and 6 days, respectively. In addition, ripening at a lower temperature (16°C) was slower with fruit fully ripened within 8 days after withdrawal from cold storage at both harvest times. ‘Reed’ avocado fruit respiration rate followed a climacteric pattern, however, significantly higher rate at higher temperature (25°C) when compared with lower temperature (16°C) after withdrawal from cold storage during both harvest times. Interestingly, mid-harvest fruit showed high electrolyte damage after withdrawal from 2.0°C when compared with late harvest fruit at the same temperature. Furthermore, mid-harvest fruit stored at 2.0ºC and ripened at 21°C showed higher chilling injury when compared with fruit ripened at 16 and 25°C. High electrolyte leakage positively correlated (R2 = 0.242) with high chilling damage for xiv mid-harvest fruit stored 5.5°C. Treatment factors had a significant effect (P < 0.05) on lightness (L *) and hue angle (h ) but no significant effect (P > 0.05) on chroma (C *) and eye colour of ‘Reed’ avocado fruit during ripening, irrespective of harvest time. Overall results showed a visual change in ‘Reed’ avocado skin colour, with eye colour changing from green to bright yellow. Furthermore, late harvest fruit showed high post-harvest pathological diseases after removal from high temperature (5.5°C) when compared with mid-harvest fruit stored at low storage temperature (2.0°C). Ripening at a higher temperature (21 and 25°C) resulted in higher incidence of stem-end rot and body rot when compared with lower temperature (16°C) for both harvest times. Late harvest fruit showed a higher incidence of vascular browning at higher ripening temperatures (21 and 25°C) when compared with lower temperature (16°C) after withdrawal from cold storage. Moreover, overall results showed that harvest time, post-harvest storage and the ripening temperature had a profound influence on the quality of ‘Reed’ avocado fruit. In conclusion, ‘Reed’ avocado fruit can be harvested during mid- or late season and stored at recommended low temperature (2.0ºC); and thereafter, ripened at either 16 or 21ºC. In addition, future studies should focus on identifying pre-harvest practices that promote higher post-harvest fruit quality for ‘Reed’ avocado fruit under South African production environment. Keywords: ‘Reed’ avocado fruit; firmness; electrolyte leakage; respiration rate; stem-end rot; body rot; vascular browning

Der SC Motor/FC Carl Zeiss Jena war seit Ende der 50-er Jahre bis in die 80-er Jahre hinein ein vom DFV der DDR und vom DTSB immer wieder benannter und bestätigter Schwerpunktclub innerhalb der sogenannten zivilen Clubs. Der SC Turbine/FC Rot-Weiß Erfurt konnte diesen Status innerhalb des Fußballverbands dagegen nie erreichen. Die zentrale Frage dieser Dissertation nach den spezifischen Bedingungsgefügen des zivilen Schwerpunktclubs FC Carl Zeiss Jena (und Vorgänger) und des zivilen Nichtschwerpunktclubs FC Rot-Weiß Erfurt (und Vorgänger) im DDR-Fußballsystem ergab sich aus dieser unterschiedlichen Privilegierung und den ungleichen Erfolgsbilanzen dieser beiden Clubs. Die Hypothese der komparativ angelegten Fallstudie vermutete einen unmittelbaren Zusammenhang zwischen diesen deutlich sichtbaren Erfolgsunterschieden der beiden Mannschaften in der DDR und den erfolgten Schwerpunktfestlegungen. Zusätzlich konnte vermutet werden, dass ein beträchtlicher Anteil an den Jenaer Erfolgen auf die besonders starke Unterstützung des wirtschaftlich mächtigen VEB Carl Zeiss Jena zurückzuführen war. Um diesen Zusammenhängen nachzugehen, fragte der Autor nach den konkreten Bevorzugungen des Jenaer Schwerpunktclubs und den Benachteiligungen des Erfurter Nichtschwerpunktclubs und nach den spezifischen Bedingungen und Handlungsspielräumen der beiden Thüringer Mannschaften in der DDR. Daraus ergaben sich eine Reihe von detaillierten, auf einen Vergleich der verschiedenen Bedingungen in Erfurt und in Jena hin orientierte, Fragen, welche in der vorliegenden Untersuchung detailliert beantwortet werden: Wie sah die besondere Förderung des DFV bzw. des DTSB für einen Schwerpunktclub wie Jena überhaupt aus? Wer nahm Einfluss auf die Clubs, von wem waren diese abhängig, wer förderte sie durch welche Leistungen? Wie wurden diese Beschlüsse vor Ort umgesetzt? Wer waren die Trägerbetriebe und in welchem Maße und wodurch engagierten sich diese für den Fußball in Erfurt und Jena? Wie kamen die häufigen Wechsel der besten Spieler Erfurts nach Jena zustande? Warum war die Richtung dieser Wechsel insgesamt einseitig in Richtung Jena? Welche finanziellen, materiellen und sozialen Bedingungen konnten den Spielern in Jena und Erfurt geboten werden? Die vorliegenden Ergebnisse dieser erstmals für die zivilen Clubs auf der Mikroperspektive angelegten systematischen Untersuchung bestätigen das bereits von Hans Joachim Teichler als grundlegend für den DDR-Fußball beschriebene Konfliktmuster des „Fußball-Lokalpatriotismus versus Parteiräson“. Eigenmächtige Handlungen vieler Betriebsleiter und zahlreicher Partei- und Gewerkschaftsfunktionäre in den Trägerbetrieben konnten beispielsweise in Erfurt bei der eigenmächtigen Erhöhung der Aufnahmezahlen von Fußballern an die KJS Erfurt oder in Jena bei der Anstellung der Fußballer im Zeisswerk nachgewiesen werden. Das am sowjetischen Vorbild orientierte Sportsystem der DDR mit seinen engen Bindungen an die Trägerbetriebe provozierte geradezu verdeckte Zuwendungen der Betriebe, die über die Clubs an die Spieler weitergereicht wurden. Für die zentralen Instanzen des DDR-Fußballs war das ein Dauerproblem, weil sich damit ein Großteil der Vorgänge vor Ort der Steuerung entzog. Wie in der vorliegenden Arbeit beschrieben wird, war genau dies jedoch der Schlüssel für den Erfolg des SC Motor/FC Carl Zeiss Jena vom Ende der 50-er bis in den Anfang der 80-er Jahre bzw. für den vergleichsweisen Misserfolg des SC Turbine/FC Rot-Weiß Erfurt im gleichen Zeitraum. Dass letztlich die finanziellen, materiellen und sozialen Möglichkeiten die entscheidende Gründe für die Spieler waren, zu einem anderen Club oder einer BSG zu wechseln, mithin demnach Marktmechanismen, und hier in erster Linie der Grund für die Stärke des SC Motor/FC Carl Zeiss Jena zu suchen ist, ist eine zentrale Erkenntnis dieser Arbeit. / The SC Motor/FC Carl Zeiss Jena was in the late 50's to the 80's one of the DFV der DDR and of the DTSB repeatedly nominated and confirmed priority club within the so-called civilian clubs. The SC Turbine/FC Rot-Weiss Erfurt could never reach this status within the Football Association. The central question of this thesis to the specific structure of conditions of the civilian priority club FC Carl Zeiss Jena (and earlier) and of the civilian non-focal clubs FC Rot-Weiss Erfurt (and earlier) in the East German football system resulted from these different privileges, and the uneven track records of these two clubs. The hypothesis of the comparative case study to suspected a direct relationship between these highly visible success differences between the two teams in the East and made the key requirements. Additionally it was suggested that a significant proportion of the Jena successes to the particularly strong support of the economically powerful VEB Carl Zeiss Jena was due. To investigate these relationships, the author asked about the specific preferences of the Jena focal club and the disadvantages of the Erfurt non-focal clubs and on the specific conditions and scope for action of the two Thuringian teams in the GDR. This resulted in a series of detailed, based on a comparison of the different conditions in Erfurt and Jena out questions that are answered in detail in this study: What was the specific support of the DFV or the DTSB for a priority club like Jena at all from? Who took effect on the club, by whom they were dependent on who they supported by what is included? How these decisions were implemented on site? Who were the carrier companies and to what extent and how dedicated these for football in Erfurt and Jena? How did the frequent change of the best players reached Erfurt in Jena? Why was the overall direction of these changes unilaterally in the direction of Jena? What financial, material and social conditions were the players in Jena and Erfurt are offered? The present results of this first time to the civilian clubs at the micro-perspective scale systematic study to confirm the already by Hans Joachim Teichler as fundamental to the East German football described patterns of conflict of "Football local patriotism versus party argue." Unauthorized actions of many managers and many party and union officials in the support operations for example, could be detected in Erfurt at the arbitrary increase in the numbers of pictures of football players at the KJS Erfurt in Jena or to the appointing of the footballers in the Zeiss factory. The Soviet model based on the GDR sports system provoked by his close ties to the carrier companies almost hidden benefits of companies that have been passed down through the clubs to the players. For the central authorities of the East German football was a constant problem because it deprived a large part of the operations of local control. As described in the present work, exactly that was, however, the key to the success of the SC Motor/FC Carl Zeiss Jena of the late 50's until the early 80's and for the comparative failure of the SC Turbine/FC Rot-Weiss Erfurt in the same period. That were the financial, physical and social opportunities the key reason for the players, ultimately, to move to another club or BSG, consequently, therefore market mechanisms, and this is primarily the reason for the strength of the SC Motor/FC Carl Zeiss Jena looking for is is a central finding of this study.

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