Dissertations / Theses on the topic 'Virus diseases of maize'

Bibliography: pages 175-184.
The occurrence of maize dwarf mosaic virus (MDMV) in field grown maize was investigated. For this purpose, maize showing mosiac symptoms was collected from different maize growing areas in South Africa by Prof. M.B. von Wechmar. These samples from Transvaal, Orange Free State and Natal were then investigated for the presence of MDMV and possible strains of this virus. Three virus isolates were purified and partially characterised. These isolates were serologically compared together with a fourth isolate SCMV 4975, obtained from the U.S., to establish strain relationships.

Responses from pedigree selection for resistance to gibberella ear rot were assessed in four maize (Zea mays L.) populations, two selected after inoculation of Fusarium graminearum (Schwabe) macroconidia into the silk channel and two selected after inoculation into developing kernels. Responses were significant in both populations selected for silk resistance and in one of the populations selected for kernel resistance. Selection was more effective in later generations and genetic gains were associated with among-family selection but not with within-family selection. Results obtained here indicate that responses to selection could be more efficiently obtained by applying high selection intensities in advanced generations, by managing earlier generations as bulks and by reducing the number of plants per family. In another experiment, a wide sample of Argentine maize germplasm was evaluated for silk and kernel resistance to gibberella ear rot and to fusarium ear rot (caused by F. verticillioides (Saccardo) Nirenberg [=F. moniliforme (Sheldon)]. Several entries exhibited disease resistance in comparison with local check hybrids, particularly for fusarium ear rot, the most prevalent ear rot in Argentina. Results obtained in this study suggested the presence of general mechanisms controlling silk and kernel resistance to both diseases. In a supplementary study, viral diseases were surveyed in maize fields from the provinces of Ontario and Quebec in 1999 and 2000. Barley yellow dwarf was found in 1999. Sugarcane mosaic, maize dwarf mosaic and wheat streak mosaic were found in 2000. These diseases were not important for grain-maize planted in May, the most prevalent kind of maize crop in these provinces. Some of these diseases, such as sugarcane maize mosaic and maize dwarf mosaic were found important only in maize fields planted during or after the month of June, and this is of commercial relevance only for sweet corn.

Gray leaf spot, caused by Cercospora zeae-maydis, can be a yield-limiting factor in maize where continuous minimum tillage practices are followed. Commercial corn hybrids were evaluated for response to gray leaf spot for seven years at two Virginia locations (Shenandoah and Wythe Counties) and one year at a third location in Virginia (Montgomery County). Yield losses, when comparing resistant to susceptible classes, were approximately 2,000 kg ha⁻¹ at Wythe County in 1982, 750 kg ha⁻¹ at Shenandoah County in 1984, and 2,150 kg ha⁻¹ at Montgomery County in 1988. The inheritance of reaction to gray leaf spot was studied using a 14 inbred diallel in Montgomery and Wythe Counties, Virginia in 1987 and 1988 planted in randomized complete block designs. Resistance was found to be highly heritable and controlled by additive gene action. Inbreds producing high yielding, resistant, and agronomically superior hybrids were identified (B68, NC250, Pa875, Va14, Va17, and Va85); and several hybrids between these lines had high levels of resistance, high yield, and good general agronomic characters (B68 x KB1250, KB1250 x Pa875, and NC250 x Pa875). Currently available inbreds could be used to produce hybrids with higher levels of resistance than hybrids currently available to growers, and these could serve as a basis for gray leaf spot breeding programs. Lesion size measurements were not correlated with disease scores. Late-season photosynthesis rates were associated positively with resistance. The hybrids of some inbreds were found to produce high levels of pigment (believed to be anthocyanins) around the gray leaf spot lesions. These did not limit the size of the individual lesion later in the season. Some pigment(s)-producing genotypes were found to be resistant when the pigment character was expressed. This type of resistance must prevent or inhibit infection of the leaf but not later colonization, once established. Maize dwarf mosaic virus (MDMV) also limits maize production in some areas where johnsongrass (Sorghum halepense L.) is a problem. Resistance to MDMV was found to be mainly additive and highly heritable. However, a strong specific combining ability component was found, indicating that the background of the material receiving resistance genes may have a strong effect on the expression of resistance. Inbreds capable of producing high-yielding, resistant, and agronomically acceptable hybrids are available (B68, NC250, A632, Pa875, Va17, and Va85); and several hybrids between these lines have high levels of resistance, high yield, and good general agronomic characters (B68 x KB1250, KB1250 x Pa875, and NC250 x Pa875).
Ph. D.

Thesis (PhD. (Geography)) --University of Limpopo, 2019
Of late climate change and consequently, the spread of crop diseases has been identified as one of the major threat to crop production and food security in subSaharan Africa. This research, therefore, aims to evaluate the role of in situ hyperspectral and new generation multispectral data in detecting maize crop viral and fungal diseases, that is maize streak virus and grey leaf spot respectively. To accomplish this objective; a comparison of two variable selection techniques (Random Forest’s Forward Variable, (FVS) and Guided Regularized Random Forest: (GRRF) was done in selecting the optimal variables that can be used in detecting maize streak virus disease using in-situ resampled hyperspectral data. The findings indicated that the GRRF model produced high classification accuracy (91.67%) whereas the FVS had a slightly lower accuracy (87.60%) based on Hymap when compared to the AISA. The results have shown that the GRRF algorithm has the potential to select compact feature sub sets, and the accuracy performance is better than that of RF’s variable selection method. Secondly, the utility of remote sensing techniques in detecting the geminivirus infected maize was evaluated in this study based on experiments in Ofcolaco, Tzaneen in South Africa. Specifically, the potential of hyperspectral data in detecting different levels of maize infected by maize streak virus (MSV) was tested based on Guided Regularized Random Forest (GRRF). The findings illustrate the strength of hyperspectral data in detecting different levels of MSV infections. Specifically, the GRRF model was able to identify the optimal bands for detecting different levels of maize streak disease in maize. These bands were allocated at 552 nm, 603 nm, 683 nm, 881 nm, and 2338 nm. This study underscores the potential of using remotely sensed data in the accurate detection of maize crop diseases such as MSV and its severity which is critical in crop monitoring to foster food security, especially in the resource-limited subSaharan Africa. The study then investigated the possibility to upscale the previous findings to space borne sensor. RapidEye data and derived vegetation indices were tested in detecting and mapping the maize streak virus. The results revealed that the use of RapidEye spectral bands in detection and mapping of maize streak virus disease yielded good classification results with an overall accuracy of 82.75%. The inclusion of RapidEye derived vegetation indices improved the classification accuracies by 3.4%. Due to the cost involved in acquiring commercial images, like xviii RapidEye, a freely available Landsat-8 data can offer a new data source that is useful for maize diseases estimation, in environments which have limited resources. This study investigated the use of Landsat 8 and vegetation indices in estimating and predicting maize infected with maize streak virus. Landsat 8 data produced an overall accuracy of 50.32%. The inclusion of vegetation indices computed from Landsat 8 sensor improved the classification accuracies by 1.29%. Overally, the findings of this study provide the necessary insight and motivation to the remote sensing community, particularly in resource-constrained regions, to shift towards embracing various indices obtained from the readily-available and affordable multispectral Landsat-8 OLI sensor. The results of the study show that the mediumresolution multispectral Landsat 8-OLI data set can be used to detect and map maize streak virus disease. This study demonstrates the invaluable potential and strength of applying the readily-available medium-resolution, Landsat-8 OLI data set, with a large swath width (185 km) in precisely detecting and mapping maize streak virus disease. The study then examined the influence of climatic, environmental and remotely sensed variables on the spread of MSV disease on the Ofcolaco maize farms in Tzaneen, South Africa. Environmental and climatic variables were integrated together with Landsat 8 derived vegetation indices to predict the probability of MSV occurrence within the Ofcolaco maize farms in Limpopo, South Africa. Correlation analysis was used to relate vegetation indices, environmental and climatic variables to incidences of maize streak virus disease. The variables used to predict the distribution of MSV were elevation, rainfall, slope, temperature, and vegetation indices. It was found that MSV disease infestation is more likely to occur on low-lying altitudes and areas with high Normalised Difference Vegetation Index (NDVI) located at an altitude ranging of 350 and 450 m.a.s.l. The suitable areas are characterized by temperatures ranging from 24°C to 25°C. The results indicate the potential of integrating Landsat 8 derived vegetation indices, environmental and climatic variables to improve the prediction of areas that are likely to be affected by MSV disease outbreaks in maize fields in semi-arid environments. After realizing the potential of remote sensing in detecting and predicting the occurrence of maize streak virus disease, the study further examined its potential in mapping the most complex disease; Grey Leaf Spot (GLS) in maize fields using WorldView-2, Quickbird, RapidEye, and Sentinel-2 resampled from hyperspectral data. To accomplish this objective, field spectra were acquired from healthy, moderate and xix severely infected maize leaves during the 2013 and 2014 growing seasons. The spectra were then resampled to four sensor spectral resolutions – namely WorldView-2, Quickbird, RapidEye, and Sentinel-2. In each case, the Random Forest algorithm was used to classify the 2013 resampled spectra to represent the three identified disease severity categories. Classification accuracy was evaluated using an independent test dataset obtained during the 2014 growing season. Results showed that Sentinel-2 achieved the highest overall accuracy (84%) and kappa value (0.76), while the WorldView-2, produced slightly lower accuracies. The 608 nm and 705nm were selected as the most valuable bands in detecting the GLS for Worldview 2, and Sentinel-2. Overall, the results imply that opportunities exist for developing operational remote sensing systems for detection of maize disease. Adoption of such remote sensing techniques is particularly valuable for minimizing crop damage, improving yield and ensuring food security.

>Magister Scientiae - MSc
Maize streak disease (MSD), caused by variants of the Maize streak virus (MSV) A strain, is the world's third and Africa’s most important maize foliar disease. Outbreaks of the disease occur frequently and in an erratic fashion across Africa and Islands in the Indian Ocean causing devastating yield losses such that the emergence, resurgence and rapid diffusion of MSV-A variants in this region presents a serious threat to maize production, farmer livelihoods and food security. To compliment current MSD management systems, a total of 689 MSV-A full genomes sampled over a 32 year period (1979-2011) from 20 countries across Africa and the adjacent Indian Ocean Islands, 286 of which were novel, were used to estimate: (i) the levels of genetic diversity using MEGA and the Sequence Demarcation Tool v1.2 (SDT); (ii) the times of occurrence and distribution of recombination using the recombination detection program (RDP v.4) and the genetic algorithm for recombination detection (GARD); (iii) selection pressure on codon positions using PARRIS and FUBAR methods implemented on the DATAMONKEY web server; (iv) reconstruct the history of spatio-temporal diffusion for MSV-A using the discrete phylogeographic models implemented in BEAST v1.8.1; (v) characterize source-sink dynamics and identify predictor variables driving MSV-A dispersal using the generalized linear models, again implemented in BEAST v1.8.1. Isolates used displayed low levels of genetic diversity (0.017 mean pairwise distance and ≥ 98% nucleotide sequence identities), and a well-structured geographical distribution where all of the 233 novel isolates clustered together with the -A1 strains. A total of 34 MSV inter-strain recombination events and 33 MSV-A intra-strain recombination events, 15 of which have not been reported in previous analyses (Owor et al., 2007, Varsani et al., 2008 and Monjane et al., 2011), were detected. The majority of intra-strain MSV-A recombination events detected were inferred to have occurred within the last six decades, the oldest and most conserved of these being events 19, 26 and 28 whereas the most recent events were 8, 16, 17, 21, 23, and 29. Intra-strain recombination events 20, 25 and 33, were widely distributed amongst East African MSV-A samples, whereas events 16, 21 and 23, occurred more frequently within West African MSV-A samples. Events 1, 4, 8, 10, 14, 17, 19, 22, 24, 25, 26, 28, and 29 were more widely distributed across East, West and Southern Africa and the adjacent Indian Ocean Islands. Whereas codon positions 12 and 19 within motif I in the coat protein transcript, and four out of the seven codon positions (147, 166, 195, 203, 242, 262, 267) in the Rep transcript (codons 195 and 203 in the Rb motif and codons 262 and 267 in site B of motif IV), evolved under strong positive selection pressure, those in the movement protein (MP) and RepA protein encoding genes evolved neutrally and under negative selection pressure respectively. Phylogeographic analyses revealed that MSV-A first emerged in Zimbabwe around 1938 (95% HPD 1904 - 1956), and its dispersal across Africa and the adjacent Indian Ocean Islands was achieved through approximately 34 migration events, 19 of which were statistically supported using Bayes factor (BF) tests. The higher than previously reported mean nucleotide substitution rate [9.922 × 10-4 (95% HPD 8.54 × 10-4 to 1.1317 × 10-3) substitutions per site per year)] for the full genome recombination-free MSV-A dataset H estimated was possibly a result of high nucleotide substitution rates being conserved among geminiviruses such as MSV as previously suggested. Persistence of MSV-A was highest in source locations that include Zimbabwe, followed by South Africa, Uganda, and Kenya. These locations were characterized by high average annual precipitation; moderately high average annual temperatures; high seasonal changes; high maize yield; high prevalence of undernourishment; low trade imports and exports; high GDP per capita; low vector control pesticide usage; high percentage forest land area; low percentage arable land; high population densities, and were in close proximity to sink locations. Dispersal of MSV-A was frequent between locations that received high average annual rainfall, had high percentage forest land area, occupied high latitudes and experienced similar climatic seasons, had high GDP per capita and had balanced maize import to export ratios, and were in close geographical proximity.
National Research Foundation (NRF), the Poliomyelitis Research Foundation (PRF), and the Thuthuka Board

Bibliography : pages 218-230.
Maize plants collected in three geographically distinct regions of South Africa were found to be doubly infected with maize dwarf mosaic (MDMV) and cucumber mosaic virus (CMV). A mixed infection of these two viruses could be maintained in maize plants grown under laboratory conditions. The possibility of synergism or of an interference mechanism between MDMV and CMV in dual infections was investigated and it was found that prior infection with CMV interfered with subsequent infection by MDMV. MDMV and CMV were shown to be non-persistently transmitted by Myzus persicae, Rhopalosiphum padi and Rhopalosipbum maidis aphids. Protoplasts were isolated from maize seedlings and could be viably maintained for up to 66 hours. The maize protoplasts were infected with CMV and MDMV either singly, or together as a mixed inoculum. Infection curves for each virus were plotted. The presence of CMV in a mixed inoculum appeared to prevent infection of the protoplasts by MDMV. Protoplasts were isolated from plants systemically infected with CMV and/or MDMV. Superinfection of protoplasts prepared from CMV infected seedlings with MDMV was not possible. As a possible vehicle for virus infection of protoplasts liposomes were produced. Initially fluorescent dyes were incorporated in them. These were fused to the maize protoplasts. Attempts were made to encapsulate virus particles in the liposomes and fuse them to maize protoplasts but this was not successful.

Zea mays was first introduced to Africa in Ghana by Portuguese traders in the 16th century. The steady spread of maize cultivation since then has made it the most important cereal crop in Africa today. Whereas improved maize genotypes and agricultural techniques enable yearly yields above 10 tons hectare-1 in the developed world, yearly yields across Africa have remained low at about 1 ton hectare-1 in most countries. Although outmoded agricultural practices are the main reason for poor yields, maize pathogens inflict substantial additional losses. Of the many pathogens currently confronting maize farmers in Africa, Maize streak virus (MSV) is the most significant.

Virus diseases of cotton have historically been of only sporadic importance to global cotton production. Recent devastating epidemics in Pakistan and other areas have brought new awareness to the potential for disaster of a pathogen once considered to be of a minor importance. Under changing conditions this pathogen (cotton leaf curl virus) has emerged as a serious problem in Pakistan and India. Cotton leaf curl virus does not occur in the United States or the rest of the western hemisphere but recent experience worldwide is a reminder that pathogens, such as this geminivirus, can be moved easily from one part of the world to another and therefor we need to be aware of the potential impact of such pathogens on local crops.

Includes bibliographical references.
Geminiviruses replicate via a rolling circle mechanism, which initiates at the origin of replication located within the long intergenic region (LIR). The viral replication associated-protein (Rep) in conjunction with the host's DNA replication machinery is responsible for the initiation and termination of the replication cycle from a stem-loop structure, located within the LIR and conserved throughout the three genera of Geminiviridae. The specific interaction between the Rep protein with sequences within the intergenic region has been well characterised for the begomoviruses and to some extent the curtoviruses; however, this interaction in the mastreviruses, and in particular maize streak virus (MSV), has yet to be fully explored. A theoretical model has been proposed based on sequence data and informed by the current understanding of replication specificity in begomoviruses. Due to the lack of conservation of the stem sequence of the stem-loop structure amongst mastreviruses, the model implicates a pair of nucleotide sequence repeats called iterons. These are located within the stem structure, and on the complementary sense side of the LIR. The former is the putative site of Rep interaction with the LIR. These iterons would therefore potentially act as the determinants of replication specificity amongst mastreviruses.

Modern agriculture is dependent on both global supply chains and crop monocultures. These features aid the evolution and spread of novel plant pathogens. Limited genetic diversity in commercial crop lines can result in widespread susceptibility to emerging pathogens. Pathogen resistance may be developed through conventional breeding approaches, or a number of transgenic strategies. This thesis focuses on the characterisation of an emerging maize disease, Maize lethal necrosis (MLN), and engineering resistant maize lines using an artificial microRNA (amiRNA) approach. MLN is a synergistic viral disease caused by the interaction of Maize chlorotic mottle virus (MCMV) with any maize-infecting member of the potyviridae. I used next-generation RNA sequencing to characterise the MLN outbreak in East Africa, discovering that local and Chinese strains of the potyvirus Sugarcane mosaic virus (SCMV) typically coinfect with MCMV. A first global MCMV phylogeny was constructed using these samples combined with new Sanger sequencing of samples in Ecuador and Hawaii. The phylogeny supported previous hypotheses of a link between the Chinese and African outbreaks, and suggested a novel link between the Hawaiian and Ecuadorian outbreaks. The SCMV sequences generated demonstrated strong evidence of extensive recombination, in line with previous reports on SCMV and potyviruses. These data also produced first reports of a number of RNA viruses in East Africa, and five novel viral-like sequences, with their presence confirmed by RT-PCR. RNA silencing is an important component of the plant immune response to viral infection. amiRNAs can be used to generate specific and effective viral resistance through Watson- Crick base pairing between the amiRNA and the (RNA) viral genome. Previous amiRNA approaches have targeted invariable genomic regions using consensus sequences. However, the high mutation rate of RNA viruses means single cells contain a variety of mutant genomes, collectively called a quasispecies. To deter the evolution of resistance breaking I devised a novel strategy to include intra-sample variation from NGS data in amiRNA design, and constructs, each containing five of these amiRNAs, were transformed into tropical maize lines. RNA silencing may be hampered by the expression of viral suppressors of silencing (VSRs). Local VSR assays demonstrated that there are no local VSRs in the MCMV genome, while systemic VSR assays showed a possible systemic VSR role for the unique P32 protein, and an interesting link between photoperiod and systemic silencing more generally.

Les virus sont considérés comme l'un des problèmes majeurs de la production du mais en Afrique Tropicale, en particulier le maize streak virus ( M. S. V. ) et dans une moindre mesure, le maize stripe virus et le maize mosaic virus. Après purification d'isolats réunionnais et production de sérum, une méthode de diagnostic fiable et rapide des 3 maladies virales a été mise au point, pour les besoins de la sélection, à l'aide d'une technique ELISA. Trois sérotypes de M. S. V. Ont été identifiés avec 5 anticorps monoclonaux un sérotype a été mis en évidence sur tous les échantillons de mais analysés, originaires de 11 pays, et sur certains de canne à sucre et poacées. Les 2 autres ont été détectés sur canne à sucre mais non sur mais. Le sérotype trouvé sur mais est transmis par Cicadulina mbila. Cette cicadelle peut être vectrice à des taux de virus inférieurs à 0,15 ng par insecte. Elle est capable d'en accumuler plus de 6 ng en un mois d'alimentation sur plante virosée. Par contre, après acquisition, la dégradation du virus est lente. Ces résultats permettent d’expliquer la bonne persistance du pouvoir infectieux en absence de multiplication virale. La tolérance de la variété composite IRAT 297 est liée à une résistance à la multiplication du virus et on à la limitation de son déplacement dans la plante. La progression de la maladie dans les feuilles est directement liée à la croissance foliaire. Par ailleurs, le virus n'est jamais détecté dans les limbes inoculés. Ces résultats suggèrent le développement d'une résistance des cellules des feuilles après leur différenciation. Plus une plante n’est âgée au moment de son infection, Moins elle en sera affectée. I 1 a été démontré que le suivi des symptômes de la virose sur feuille permet de sélectionner la résistance à la multiplication virale. Une relation a été établie entre cette résistance et une échelle de notation qui permet de cribler les variétés tolérantes d'après leurs symptômes. Cette tolérance peut être précisée par des dosages d'antigènes viraux mais la relation entre taux de virus et rendement reste à établir.

Maize (Zea mays L.) is a cereal crop grown throughout the world. It plays an important role in the diet of millions of African people due to its high yields per hectare, its ease of cultivation and adaptability to different areas, its versatile food uses and storage characteristics (Asiedu, 1989). Maize is a staple crop in Southern Africa where it accounts for 70% of total human intake of calories (Martin et al., 2000). Thus it is essential that maize can be sustainably produced in South Africa and that maize seeds are of the highest possible quality. Fungi rank as the second biggest cause of deterioration and loss of maize (Ominski et al., 1994). At the very early stages of seedling development, maize seedlings are attacked by fungi such as Pythium, Fusarium and Rhizoctonia spp., which cause severe diseases, including pre-emergence damping-off, which lead to yield losses (Dodd & White, 1999). These diseases can be effectively controlled by applying fungicidal seed treatments (Peltier et al., 2010). However, these seed treatments should be tested to ensure that they provide an acceptable level of control against the pathogens and that they do not have any negative effects on the germination and vigour of the maize seed. In Chapter 3 of this dissertation, three important fungal genera, namely Pythium, Fusarium and Rhizoctonia spp., were isolated from diseased maize plant samples and soil. The beet seed baiting method was used for Rhizoctonia sp. and the citrus leaf disk baiting method for Pythium sp. Fusarium sp. was isolated by means of serial dilution on a selective medium. The selective media used were agar containing chlorotetracycline hydrochloride and streptomycin sulfate for Rhizoctonia, pimaricin and vancomycin, PARP (pimaricin + ampicillin + rifampicin + pentachloronitrobenzene (PCNB) agar) for Pythium sp. and Rose Bengal Glyceraldehyde Urea (RBGU) for Fusarium sp. These fungal isolates, as well as some isolates revivedfrom the University of Pretoria’s culture collection and obtained from the Agricultural Research Council (ARC-PPRI), were used for pathogenicity trials conducted on maize in the between-paper method (BP), and in six-celled plastic seedling trays in the greenhouse (described in Chapter 5). In order to test the efficacy of Stamina, Flite and Celest® XL for controlling Pythium spp., Fusarium spp. and Rhizoctonia spp. in vitro, each of the three fungicides was added to PDA at concentrations of 1, 2 and 3ppm. In order to mirror the treatments used in other experiments, a combination of Stamina and Flite was also incorporatedinto PDA at concentrations of 1, 2 and 3ppm each. A 5mm2 block of each of the fungi was plated onto the centre of the media and incubated at 25C. The diameter of the fugal growth was measured at regular intervals depending on the rate of growth of the fungus. It was found that Celest® XL was very effective in controlling all three of these pathogens in vitro, confirming research done by Govender (2005), who found that Celest® XL effectively controlled these pathogens on maize. The combination of Stamina and Flite also controlled these pathogens although to a lesser extent. Research done by BASF in 2008 showed that Stamina is able to control Pythium, Fusarium and Rhizoctonia spp. Pyraclostrobin (the active ingredient of Stamina) has also been found to effectively control all three of these pathogens in numerous in vitro and in vivo experiments (Broders et al., 2007; Peltier et al., 2010; Solorzano & Malvick, 2011). In Chapter 4 of this dissertation, the effect of three different fungicides (Stamina, Flite and Celest® XL) on the germination and vigour of two Zea mays cultivars (Monsanto DKC78-15B and PANNAR 6Q308B) was assessed. This was achieved by carrying out a standard germination test, a cold soil test, short accelerated ageing and long-term storage tests according to the guidelines of the International Seed Testing Association (ISTA, 2012). It was found that none of the fungicides had a detrimental effect on either seed germination or vigour and no phytotoxic effects were observed. The combination of Stamina and Flite treatment also led to an increased percentage germination after the cold soil test when compared to the untreated control. This confirms the research of Govender (2005), who showed that Celest® XL had no negative effects on the germination or vigour of maize, and BASF (2008), which showed that Stamina could even lead to increased germination and an increased yield of maize under cold conditions when compared to an untreated control. Bradley et al. (2001) found that fungicide seed treatments do not affect the vigour and viability of maize seeds. Seeds treated with fludioxonil also showed an increased radicle length in some cases (Munkvold & O’Mara, 2002). Increased radicle length could indicate increased vigour of the seeds (Matthews & Khajeh-Hosseini,2006).
Dissertation (MSc (Agric))--University of Pretoria, 2013.
Microbiology and Plant Pathology
MSc (Agric)
Unrestricted

Avian leukosis virus (ALV)-induced osteopetrosis is a proliferative disorder of the bone affecting the growth and differentiation of osteoblasts. Osteopetrosis is a polyclonal disease in which cells of the bone contain, on average, multiple viral DNA copies. Osteopetrotic bone is also characterized by the accumulation of unintegrated viral DNA, suggesting an atypical life cycle of the virus in the infected osteoblasts. To better understand virus-host interactions in the induction of osteopetrosis by ALVs, infected chick osteoblast cultures and osteopetrotic bone were examined for aspects of the virus life cycle and effects of infection on osteoblast function. Levels of infection and virus expression were compared in cultured osteoblasts and osteopetrotic bone. Osteopetrotic bone contained higher levels of viral DNA and correspondingly higher levels of viral proteins than infected osteoblast cultures, suggesting a higher viral load in the diseased bone. A significant level of mature Gag protein was present in the bone, suggesting the accumulation of mature virus particles in the diseased bone. It is possible that the accumulation of virus could facilitate the high levels of infection observed in the diseased bone. The mechanism by which unintegrated viral DNA persisted in osteopetrotic bone was investigated by examining the susceptibility of infected osteoblasts to superinfection. The results indicated that, in culture, infected osteoblasts were able to establish interference to superinfection. This suggests that the persistence of unintegrated viral DNA in osteopetrotic bone may not result from the continuing infection of productively infected osteoblasts. The effect of virus infection on osteoblast function was examined in the diseased bone and in osteoblast cultures. In infected chickens, osteoblast activity, as evidenced by the expression of osteoblast phenotypic markers, was increased only in chickens developing severe osteopetrosis. In culture, virus infection had no apparent effect on either the proliferation or differentiation of osteoblasts. This indicates that infection was itself not sufficient to perturb osteoblast function. Furthermore, it suggested that additional components of the bone may be required for ALV infection to induce the abnormal activity of osteoblasts observed in osteopetrosis.

Bibliography: pages 190-214.
This thesis describes investigations into the potential use of the Subgroup I geminivirus, maize streak virus (MSV), as a gene vector. These involved testing MSV-based replicons in transgenic cell lines, in transient expression assays in maize cells and in an infectious gene expression system in plants. MSV vectors which contained three different versions of a bar (bialaphos resistance) expression cassette in place of the viral movement and coat protein genes were used to generate transformed maize cell lines. A high proportion of these contained MSV-based episomes at high copy number. However, embryogenic maize tissue of the Hill line was not regenerable when an MSV-based replicon was present, possibly due to toxicity of the viral replication associated protein. In non-regenerable Black Mexican sweetcorn cell lines some of the MSV-bar episomes, which ranged in size from 3.15 kb to 4.78 kb, replicated for periods of over two years, and appeared structurally stable. The cellular levels of the bar gene product, phosphinothricin acetyl transferase (PAT), were significantly enhanced in lines where the gene was amplified by linkage to an MSV replicon in comparison with lines where the same gene was not amplified. Northern blot analysis also showed that higher levels of bar mRNA were produced in lines where the gene was amplified. However, the 3- to 5-fold enhancement in gene expression was less than was anticipated, based on results from similar Subgroup ill geminivirus-based transgene amplification systems. Several mutants of the MSV genome were generated to investigate the extent to which genome amplification contributes to the expression of the viral coat protein gene. The introduction of an Ncol restriction site at the start of the coat protein gene facilitated fusion of the gus marker gene with the coat protein upstream transcription and translation regulatory sequences. In one viral construct the plus strand origin of replication was inactivated by insertion of a short oligonucleotide; in another, the viral rep gene was inactivated by a frameshift mutation. These constructs were used to show that the MSV coat protein promoter has low, but measurable constitutive activity in the absence of genome amplification, but that viral replication enhances coat protein expression about 45-fold. I found no evidence for Rep-mediated transactivation of the coat protein promoter.

Includes bibliographical references.
Maize streak virus (MSV), circular single stranded DNA (ssDNA) virus (~2.7kb), is the causative agent of Maize streak disease, and is a devastating pathogen that causes severe crop losses to subsistence farmers in sub-Saharan Africa. MSV is transmitted by the leafhopper Cicadulina mbila, and is the type member of the Mastrevirus genus (family Geminiviridae). MSV shares a unique twinned icosahedral ("geminate") virion architecture (22 x 38 nm) with all other family members. Geminate particles consist of 110 coat protein (CP) subunits that assemble onto a circular single-stranded DNA (ssDNA) genome. Each T= I unit is an incomplete icosahedron assembled from 55 CPs. The structures of MSV and African cassava mosaic virus (ACMV, genus Begomovirus) have been studied by electron cryo- microscopy (cryo-EM) previously. While these investigations revealed some details about the geminate architecture, the interactions of capsid components have not yet been adequately modelled. The two incomplete icosahedral "heads" of the geminate particle are offset from one another and apparently make distinct CP:CP contacts at this region of the virion. Information regarding the nature of quasi- equivalent CP conformers or the sets of amino acid residues that mediate these interactions has not been forthcoming. Since the experimental results of these previous studies are not available in a public database, we were motivated to revisit the structure of MSV in order to obtain a 3D experimental density that might aid pseudo-atomic modelling. The MSV CP:ssDNA interaction has also been shown to be crucial for systemic movement through the host. Hence, quasi-atomic modelling may inform development of antiviral strategies which aim to interfere with virion assembly. MSV virions were isolated from the leaves of maize plants infected by agro-inoculation and visualized in both heavy metal stain and vitreous ice after they had been adsorbed to a thin-layer of continuous carbon to prevent virion aggregation. Virus preparations consisted of distinct CP assemblies consisting of multiples of the incomplete T=I icosahedral unit. Monopartite (icosahedral), bipartite (geminate), tripartite, and higher assemblies were observed suggesting the MSV CP is not only multifunctional but also structurally versatile being able to package ssDNA of variable sizes. Low-dose images were recorded on film, and 3D reconstruction of both monopartite and bipartite capsid species carried out using standard single-particle image processing methodology. The resolution of the bipartite reconstructions was 26 A for the negative-stain dataset, and 23 A for cryo-EM dataset, while the resolution of the monopartite reconstruction was estimated to be ~15 A. Comparative modelling of the MSV CP was undertaken using the pentamer (CPs) of Satellite tobacco necrosis virus (STNV) as a structural template. Correlation-based fitting of icosahedral and geminate atomic models that varied in geometric arrangement of MSV CPs allowed the geometric parameters of the bipartite capsid to be determined. Fitting ofMSV CPs into the EM densities informed our understanding of interfaces which allow the CP to self-associate, and showed that CPs is in fact displaced within the icosahedral geometry of the heads by a 10° rotation about the 5-fold axes of symmetry in comparison to STNV; hence, while quaternary structure of the pentameric capsomer is conserved between these viruses, the quaternary interactions between capsomers of the T=I unit has diverged considerably. This study shows that the offset between the geminate heads of the MSV virion is ~-11°, and that this geometry appears to arise owing to a distinct set of CP:CP interfaces which occur across the equator between two quasi-icosahedral heads and involve regions that would interact to form the CPs: CPs interfaces within each of the heads (across 2-fold and 3-fold symmetry axes). Notably this offset differs from that reported for ACMV, which has a reported offset of 20°. Additionally, the resolution afforded by the icosahedral monopartite reconstruction provided the first structural evidence to suggest that the calcium ion binding site of the STNV CPs (located on the CS axis) is likely to be conserved in MSV. This result suggests that in common with other plant viruses, depletion of calcium ions may be required for genome egress in a newly infected host cell. This study highlights the importance of future high-resolution studies of this unique virion morphology by both X-ray crystallography and cryo-EM.

La demanda mundial d’aliments ha dut al desenvolupament d’una intensa activitat agrícola produint la fragmentació, modificació i simplificació dels hàbitats naturals ja documentats com una de les principals causes de l’extinció i pèrdua de la diversitat d’espècies. La biodiversitat que tenen els ecosistemes proveeix d’una sèrie de serveis ecosistèmics a l’ésser humà i el control biològic natural de plagues es un dels serveis que s’ha vist alterat especialment als agroecosistemes. Tot i que existeix una creixent literatura científica que suggereix que la presència d’hàbitats naturals beneficia als enemics naturals als groecosistemes, l’eficiència del control biològic segueix sent un dels principals reptes en aquests estudis, oncs els efectes varien molt entre espècies i entre estudis. Entendre els patrons d’abundància i moviment de les espècies d’enemics naturals i d’herbívors, així com les interaccions entre aquests en els paisatges agrícoles, es espacialment i temporal complex. Si bé la gestió dels hàbitats agrícoles ofereix solucions per a reduir la pèrdua del rendiment degut a les plagues, en el cas dels virus, la simplificació excessiva de la diversitat dels cultius, els sistemes intensius de cultiu i l’ús de productes fitosanitaris interfereixen amb les funcions ecològiques dels agroecosistemes alterant la epidemiologia de malalties a les plantes. Amb l’objectiu d’oferir una perspectiva general sobre la’influència que el paisatge agrícola pot tenir sobre el control biològic i la epidemiologia viral, aquesta tesis s’ha enfocat en analitzar els efectes del paisatge agrícola des d’una perspectiva de composició espacial i del maneig del camp sobre espècies d’insectes herbívors i depredadors, així com en els principals virus que afecten al cultiu del panís a la zona de la Vall de l’Ebre. La primera part de la tesis està orientada a (1) l’ampliació en el coneixement de quins són els tipus d’estudis que s’estan duent a terme per analitzar els efectes de l’agroecosistema en el control biològic, les diferents metodologies i els resultats més recents (Capítol 1), i (2) els diferents efectes que té l’estructura (composició i configuració) del paisatge i les variables locals en les espècies de depredadors i herbívors presents en el cultiu de panís (Capítol 2). La segona part de la Tesi està orientada a estudiar els efectes del paisatge sobre tres dels principals virus que afecten al cultiu de panís (1) dos virus generalistes de vector: el virus del mosaic de la canya de sucre (SCMV) i el virus del mosaic nanitzant del panís (MDMV) (Capítol 3), i un virus especialista de vector: el virus del nanisme rugós del panís (MRDV) (Capítol 4). Les principals conclusions d’aquesta tesis doctoral són: (1) els estudis de caràcter funcional on s’estudia composició del paisatge han demostrat que les espècies d’enemics naturals necessiten hàbitats specífics, però que altres variables com són els efectes locals del cultiu també poden afectar a les seves dinàmiques i les de les plagues amb més força; (2) la superfície de fruiters afecta negativament als depredadors potencials del panís, però que les cobertes vegetals d’aquests afecten positivament a certes espècies d’herbívors en absència del panís; (3) l’alfals actua com un cultiu que aporta depredadors i herbívors al cultiu de panís actuant com un recurs per a les espècies més importants dels hàbitats seminaturals; (4) la incidència viral al panís està influïda principalment per la data de sembra i per la cobertura i presència d’espècies de gramínies als marges. Aquests resultats obren una via en el coneixement dels efectes que produeixen tant la composició del paisatge agrícola com el maneig del propi camp en l’abundància i distribució d’herbívors, depredadors i de virus en el cultiu de panís. Aquests resultats a més suggereixen que s’han de contemplar mesures de gestió a escala local que garanteixin nivells mínims de biodiversitat i sostenibilitat com a base inicial per al desenvolupament de programes més eficients de control biològic de plagues i virus.
La demanda mundial de alimentos ha llevado al desarrollo de una intensa actividad agrícola produciendo una fragmentación, modificación y simplificación de los hábitats naturales ya documentada como una de las principales causas de la extinción y pérdida de la diversidad de especies. La cantidad, estructura y forma de la superficie de estos hábitats naturales fragmentados genera efectos positivos o negativos sobre la abundancia y diversidad de las especies. La biodiversidad que tienen los ecosistemas provee de una serie de servicios ecosistémicos al ser humano, y el control biológico natural de plagas es uno de los servicios que se ha visto alterado especialmente en los agroecosistemas. Aunque existe una creciente literatura científica que sugiere que la presencia de hábitats naturales beneficia a los enemigos naturales en los agroecosistemas, la eficiencia del control biológico sigue siendo uno de los principales retos en estos estudios, pues los efectos varían mucho entre especies y entre estudios. Entender los patrones de abundancia y movimiento de las especies de enemigos naturales y de herbívoros en los paisajes agrícolas, así como las interacciones entre estos, es espacial y temporalmente complejo. Si bien la gestión de los hábitats agrícolas ofrece soluciones para reducir la pérdida de rendimiento debido a las plagas, en el caso de los virus la simplificación excesiva de la diversidad de los cultivos, los sistemas intensivos de cultivo y el uso de productos fitosanitarios interfieren con las funciones ecológicas de los agroecosistemas alterando la epidemiología de enfermedades en las plantas. Con el objetivo de ofrecer una perspectiva general sobre la influencia que el paisaje agrícola puede tener sobre el control biológico y la epidemiología viral, esta tesis se ha centrado en analizar los efectos del paisaje agrícola desde una perspectiva de composición espacial y del manejo del campo sobre especies de insectos herbívoros y depredadores, así como en los principales virus que afectan al cultivo de maíz en la zona del Valle del Ebro. La primera parte de la tesis está orientada a (1) la ampliación en el conocimiento de cuáles son los tipos de estudios que se están realizando para analizar los efectos del agroecosistema en control biológico, las diferentes metodologías y los resultados más recientes (Capítulo 1), y (2) los efectos que tiene la estructura (composición y configuración) del paisaje y las variables locales en las especies de depredadores y herbívoros presentes en el cultivo del maíz (Capítulo 2). La segunda parte de la tesis está orientada a estudiar los efectos del paisaje sobre tres de los principales virus que afectan al cultivo del maíz (1) dos virus generalistas de vector: el virus del mosaico de la caña de azúcar (SCMV) y el virus del mosaico enanizante del maíz (MDMV) (Capítulo 3), y un virus especialista de vector: el virus del enanismo rugoso del maíz (MRDV) (Capítulo 4). Las principales conclusiones de esta tesis son: (1) los estudios de carácter funcional donde se analiza la composición del paisaje han demostrado que las especies de enemigos naturales necesitan de hábitats específicos, pero que otras variables como son los efectos locales del cultivo también pueden afectar a las dinámicas de los enemigos naturales y los herbívoros con más fuerza; (2) la superficie de frutales afecta negativamente a los depredadores potenciales en el maíz, pero que las cubiertas vegetales de estos afectan positivamente a ciertas especies de herbívoros; (3) la alfalfa actúa como un cultivo que aporta depredadores y herbívoros al cultivo del maíz actuando como un recurso para las especies con mayores efectos que los hábitats semi-naturales; (4) la incidencia viral en el maíz está influenciada principalmente por la fecha de siembra y por la cobertura y presencia de especies de gramíneas en los márgenes. Estos resultados abren una vía en el conocimiento de los efectos que produce tanto la composición del paisaje agrícola como el manejo del propio campo en la abundancia y distribución de herbívoros, depredadores y de virus en el cultivo del maíz. Además, son una base para predecir las consecuencias de la modificación de las prácticas agrícolas en el control biológico de plagas y de virus en el cultivo del maíz. Estos resultados además sugieren que deben de contemplarse medidas de gestión a escala local que garanticen niveles mínimos de biodiversidad y sostenibilidad como base inicial para el desarrollo de programas más eficientes de control biológico de plagas y virus.
The global demand for food has led to the development of intense agricultural activity resulting in fragmentation, modification and simplification of natural habitats already documented as one of the main causes of extinction and loss of species diversity. The quantity, structure and surface shape of these fragmented natural habitats generate positive or negative effects on the abundance and diversity of species. The biodiversity that ecosystems have provides a series of ecosystem services to humans, and natural biological control is one of the services that has been altered, especially in agroecosystems. Although there is growing scientific literature that suggests that the presence of natural habitats benefits natural enemies in agroecosystems, the efficiency of biological control remains one of the main challenges in these studies, as the effects vary greatly between species and between studies. Understanding the patterns of abundance and movement of natural enemies and herbivores, as well as understanding the interactions between them, is spatially and temporarily complex in agricultural landscapes. While agricultural habitat management offers solutions to reduce yield loss due to pests, in the case of viruses, the excessive simplification of crop diversity, intensive cropping systems and the use of phytosanitary products interfere with the ecological functions of agroecosystems, altering the epidemiology of diseases in plants. With the aim of offering a general perspective on the influence that the agricultural landscape can have on biological control and viral epidemiology, this thesis has focused on analysing the effects of the agricultural landscape from a spatial composition and field management perspective on species of herbivorous and predatory insects, as well as in the main viruses that affect the cultivation of maize in the area of the Ebro Valley. The first part of the thesis is aimed at (1) broadening the knowledge of identifying the types of studies that are being conducted to analyse the effects of agroecosystem on biological control, the different methodologies and the most recent results (Chapter 1), and (2) the effects of the structure (composition and configuration) of the landscape and the local variables in the predators and herbivores present in the cultivation of maize (Chapter 2). The second part of the thesis is aimed at studying the effects of the landscape on three of the main viruses that affect the cultivation of maize (1) two generalist vector viruses: the sugarcane mosaic virus (SCMV) and the maize dwarf mosaic virus (MDMV) (Chapter 3), and a vector specialist virus: the maize rough dwarf virus (MRDV) (Chapter 4). The main conclusions of this thesis are as follows: (1) functional studies where landscape composition is studied have shown that natural enemy species need specific habitats, while other variables such as the local effects of the crop can also affect the dynamics of natural enemies and herbivores with greater weight; (2) the surface of orchards negatively affects potential predators in maize, but the plant cover of these positively affects certain species of herbivores; (3) alfalfa acts as a crop that contributes predators and herbivores to the cultivation of maize acting as a resource for species with higher effects than semi-natural habitats; and (4) the viral incidence in maize is mainly influenced by the planting date and the cover and presence of grass species at the edges. These results offer additional knowledge about the effects produced by both the composition of the agricultural landscape and the management of the field itself in the abundance and distribution of herbivores, predators and viruses in the cultivation of maize. In addition, they provide a basis for predicting the consequences of the modification of agricultural practices in the biological control of pests and viruses in the cultivation of maize. These results also suggest that local management measures that guarantee minimum levels of biodiversity and sustainability should be considered as the initial basis for the development of more efficient biological control programmes for pests and viruses.

It is shown here that the ability of B lymphocytes to act as supportive host cells for virus infections requires they be activated from the resting Gostage of the cell cycle. I have used a series of activation regimens, which allow B cells to progress to different stages in their activation/differentiation pathway toward antibody secretion, in order to evaluate the extent of activation required to support vesicular stomatitis or Newcastle disease virus infections. At least three distinct phases during B cell activation which affected VSV infection were defined. Freshly isolated resting murine splenic B cells in the Go phase of the cell cycle do not support VSV, assessed by protein synthesis, infectious center formation, and PFU production. Small B cells cultured for 48 hours without stimulation still do not support VSV. B cells stimulated with the lymphokines found in Con A activated supernatants from splenic T cells or cloned T cell lines transited into the G1 phase of the cell cycle but remain refractory to VSV. These VSV non-supportive B cell populations do take up virus particles and transcribe viral mRNAs which can be translated in vitro, suggesting a translational block to VSV. B cells stimulated into the S phase of the cell cycle with anti-immunoglobulin synthesize VSV proteins and increased numbers of infectious centers, but only low level PFU synthesis (center) is observed. Co-stimulation with anti-Ig and lymphokines, which supports differentiation to antibody secretion, enhanced PFU synthesis without further increasing the number of infected B cells. LPS, which activates B cells directly to antibody secretion by a pathway different from anti-Ig, induced infectious centers, and PFUs at levels comparable to those seen when stably transformed permissive cell lines are infected. Co-stimulation of LPS activated B cells with the same lymphokine populations that enhance PFU production when anti-Ig is used as a stimulator suppresses PFU production completely, suggesting that anti-Ig and LPS activated B cells are differentially responsive to lymphokines. NDV infection of murine B cells differed markedly from VSV infection, as all B cell populations examined gave a similar response pattern. NDV viral proteins were synthesized by B cells in each of the activation states previously described, even freshly isolated B cells. Infectious center formation increased up to 5-fold over the levels observed with unstimulated B cells after anti-Ig or LPS activation. However, PFU synthesis was low (center) for all B cell populations. These results suggest that these two similar viruses may be dependent on different host cell factors and that these factors are induced for VSV but not NDV by the B cell activators employed here or that the process of infection of B cell by these two viruses induces different cellular responses.

A number of plant viruses were isolated from diseased cucurbits in Arizona during 1982 -85. Watermelon mosiac virus 2, cucumber mosaic virus, and squash mosaic virus are previously recognized viral pathogens in Arizona and in most years are not considered economically threatening to cucurbit production. Three newly described plant viruses (lettuce infectious yellows, watermelon curly mottle and zucchini yellow mosaic) however, have the potential to, or already have, incited serious epidemics in Arizona. Losses are heaviest with these diseases when insect vector levels build up early in the growing season and plants become infected during critical developmental stages.

The South African isolate of the Black Queen-Cell Virus (BQCV), a honeybee virus, was previously found to have an 8550 nucleotide genome excluding the poly(A) tail. Its genome contained two ORFs, a 5'-proximal ORF encoding a putative replicase protein and a 3'-proximal ORF encoding a capsid polyprotein.

A reverse transcriptase PCR (RT -PCR) assay was developed for the detection of BQCV and acute bee-paralysis virus (ABPV). Complete genomes sequences w ere used to design unique PCR primers within a l-kb region from the 3' end of both genomes to amplify a fragment of 70.0 bp from BQCV and 900 bp from ABPV. The combined guanidinium thiocyanate and silica membrane method was used to extract total RNA from samples of healthy and laboratory-infected bee pupae. In a blind test, RT-PCR successfully identified the samples containing BQCV and ABPV. Sensitivities were of the order of 130 genome equivalents of purified BQCV and 1600 genome equivalents of ABPV.

Bibliography: pages 199-213.
The infectivity of the replicative form (RF) DNAs of MSV-Kom, MSV-Set and PanSV-Kar contained in the plasm ids pKom500, pSet100 and pPS100 was established by agroinoculating susceptible Jubilee sweetcorn with partial homodimeric Agrobacterium tumefaciens (C58C1) clones of RF-DNAs. Biological characteristics typical of Mastreviruses; such as, the appearance and leafhopper transmissibility of streak symptoms on infected plants, the presence of 18x30nm geminate particles in electron micrographs of leaf-dip preparations, and the presence of single-stranded and double-stranded DNA in Southern blot tests of infected plant DNA extracts, indicated that the RF-DNAs in pKom500, pSet100and PanSV-Kar represent the entire genomes of MSV-Kom, MSV-Set and PanSV-Karrespectively. The complete nucleotide (nt) sequence of the genome of MSV-Set was determined and characterised, and compared with those of MSV-Kom and PanSV-Kar. The genome sizes of MSV-Kom, MSV-Set and PanSV-Kar are 2701, 2690 and 2705 nt respectively, and all share Mastreviral genomic features. Phylogenetic analyses on the nt sequences and the putative amino acid sequences of the movement, coat and replication-associated proteins (MP, CP and Rep respectively) indicate that MSV-Set is grouped with, yet distinct from the MSV group of viruses isolated from maize. MSV-Set shares a 78% nt sequence identity with MSV-Kom which shares a >96% nt sequence identity with other MSVs. The PanSV-Kar genome shares a 60% nt sequence identity with the MSV group and89% with the Kenyan PanSV-Ken. PanSV-Kar causes mild non-persistent streak in Jubilee sweetcorn. MSV-Kom (previously isolated from maize in Komatipoort, Mpumalanga) and MSV-Set (previously isolated from a Setaria species in Mt. Edgecombe, Kwazulu/Natal) have different pathogenicities, and have overlapping, but non-identical, host ranges. Leafhopper transmission tests determined that MSV-Kom and MSV-Set generally cause severe and moderate streak in maize cultivars, or mild and severe streak in wheat cultivars respectively.