Cassava brown streak disease (CBSD) caused by two distinct ssRNA virus species (CBSV and UCBSV of genus Ipomovirus, family Potyviridae) and transmitted by whitefly (Bemisia tabaci), is a major constraint to cassava production in Africa, including Mozambique. In this research, two studies were conducted. First, in order to monitor the incidence, severity and geographical distribution of cassava brown streak disease and associated viruses in Mozambique, field surveys were performed in six cassava major growing provinces. A total of one hundred and fifteen fields and one hundred and forty six fields were surveyed in 2010 and 2012, respectively. The disease was only found in three of six provinces namely Zambezia, Nampula and Cabo Delgado. The CBSD incidence was highest (61.3% and 82.2% in 2010 and 2012, respectively) in Zambezia and lowest (23.6% and 35.1% in 2010 and 2012, respectively) in Cabo Delgado, with cultivars such as Cadri and Robero showing the highest susceptibility to CBSD, while Likonde and Amwalikampiche had relatively low CBSD incidence, illustrating some tolerance to the disease. The results, when compared to previous surveys conducted in 1999 and 2003, demonstrated that the disease is increasing, and replanting new fields with disease-affected cuttings could be responsible for the spread. The second aim of the study was to investigate the genetic diversity of Cassava brown streak viruses, based on analysis of partial sequences of the coat protein gene, in Mozambique. Collections of CBSD-symptomatic leaves were done between June 2010 and June 2012. Diagnostic RT-PCR, using specific primers to screen for the two species, revealed for the first time the presence of Uganda cassava brown streak virus (UCBSV) in Mozambique. UCBSV was found in mixed infections with CBSV, and only confined to a single province of Zambézia, while CBSV species were widely distributed. The phylogenetic analysis revealed two subgroups within CBSV, which were 6.7% divergent in nucleotide sequence. The heterogeneity observed among CBSV isolates in Mozambique suggests that in the future studies more sampling is needed to characterize strains and variants. Addtionally, sequencing of the full CP sequence of CBSaVs isolates is required, which may reveal even more diversity. Infectivity assays of cassava brown streak viruses (CBSV and UCBSV) were established using the host indicator plant Nicotiana benthamiana. Plant sap was extracted from infected cassava leaves and inoculated into N.benthamiana plants. CBSD-like symptoms were observed, and RT-PCR revealed the presence of CBSV in all samples, except for one which was co-infected with UCBSV and CBSV. This study provided further evidence that CBSaVs are efficiently transmitted to N.benthamiana. There is scanty information on alternative hosts, therefore more research is needed to identify other potential hosts of CBSaVs in order to develop an effective strategy to control CBSD.

The limited specificity, sensitivity and multiplex capacity of detection techniques currently available for important seed-borne pathogens of tomato is a significant risk for the global tomato trade and production industry. These pathogens can be associated with seed at low concentrations but, due to their highly virulent nature, these low levels can be sufficient to infect germinating seedlings and spread to neighbouring plants and fields, potentially causing epidemics and economic losses. In this study, detection techniques currently available for phytodiagnostics were evaluated for the capacity to accurately detect and identify five agronomically important seed-borne pathogens of tomato: Pepino mosaic virus (PepMV), Tomato mosaic virus (ToMV), Clavibacter michiganensis subsp. michiganensis (Cmm), Xanthomonas campestris pv. vesicatoria and Pseudomonas syringae pv. tomato. A prototype diagnostic microarray was also designed in an attempt to develop a tool that could simultaneously detect these five seed-borne pathogens from a single sample. Viral detection based on serological techniques was rapid, accurate and reliable but only detected a single pathogen per assay and required supplementary bioassays to indicate the viability of detected viral pathogens. Selective media plating for bacterial detection demonstrated unreliable recovery of targeted bacteria from infected seed and leaf samples and required supplementary tests to validate the identity of presumptive positives. Assays were lengthy, laborious and sometimes too ambiguous for accurate diagnosis of bacterial pathogens. Nucleic acid-based technologies demonstrated improved sensitivity and specificity for detection of targets from pure culture, leaf and seed extracts, compared to conventional and serological methods, yet also required supplementary bioassays or media assays to validate the viability of detected pathogens. Amplification efficiency however, was affected by the presence of PCR inhibitors and despite positive detection, variable banding intensity in electrophoretic analysis of amplified products necessitated the use of reference cultures to validate diagnosis. The developed microarray incorporated 152 pathogen-specific and control probes to facilitate diagnosis and taxonomic classification of detected pathogens. The array was challenged with pure culture extracts of the five target pathogens, selected related and non-target, unrelated pathogens of tomato. Positive detection of each of the pathogens was demonstrated but the production of hybridisation signals was highly variable and extremely sensitive to minor technical differences. Each of the five pathogens were successfully detected in combination proving that different classes of seed-borne pathogens could be detected from a single sample using the developed microarray. This prototype microarray has good potential for phytodiagnostic screening of the five targeted pathogens, and further validation, optimisation and extension for testing tomato seed samples may facilitate incorporation of this array into standard diagnostic protocols.

Solanum lycopersicon (the cultivated tomato) is a commodity of great economic importance in South Africa (SA) as well as worldwide. A destructive viral disease known as Tomato curly stunt virus, ToCSV-[ZA:Ond:98], belonging to the genus Begomovirus has negatively impacted on tomato production in SA. This has brought about the need to develop resistant cultivars to ToCSV. Since all cultivated tomato cultivars are susceptible to ToCSV, resistance genes against the virus found in wild tomato plant species have been introgressed into the cultivated tomato by plant breeding techniques. Wild relatives of tomato were adapted to many pathogens (including viruses) as well as stresses from the surrounding environment. During breeding for improved fruit quality and increased yield, the gene networks giving rise to many biotic and abiotic stress resistances have been lost leaving the domesticated tomato extremely susceptible. Plant breeders have reconstituted some of the gene networks into the cultivated tomato that provide tolerance to stresses including viruses. They have achieved this by the help of marker-assisted selection (MAS), where the associated marker is used as an indirect selection criterion. This is an important process in commercial breeding programs as it allows for a speedy selection of selected traits in the development of tomato hybrids. The defence response to abiotic stresses in plants includes the expression of heat shock proteins (HSPs) that function as stress response proteins, molecular chaperones and proteases which repair or degrade damaged proteins. The objective of this study was to elucidate the type of resistance mechanism of a tomato inbred line (TAM), to ToCSV. Since TYLCV-IL shows 77% nucleotide identity with ToCSV, molecular markers already established for the detection of resistance genes for TYLCV-IL were used to screen TAM. The inbred line, TAM, was screened for the absence of any of the known resistant genes to TYLCV-IL using molecular markers already established for the screening of TYCLV-IL resistance genes. TAM was crossed with susceptible cultivar, Rooikhaki, to produce F1 hybrids. These F1 hybrids were selfed to produce an F2 population. Infection trials using ToCSV were conducted using TAM inbred line, F1 hybrids and the F2 population. Since TAM did not have any of the known resistance genes to TYLCV-IL, a possible novel resistance source to ToCSV was speculated. A clue to the resistant mechanism against ToCSV resistance in TAM was indicated by the segregation patterns of the F2 population after inoculation with ToCSV. The results suggest that the resistance is under the control of partially dominant resistant genes. The level of resistance of commercial South African tomato cultivars (Tyler and Tovi-star) against TYLCV-IL was investigated. The heat shock protein (HSP) profiles of these two SA lines including susceptible cultivar, Rooikhaki, were treated with abiotic stresses (salt and heat) and results were compared with a similar study conducted with TYCLV-IL resistant and susceptible tomato cultivars. Heat shock protein 70 accumulation patterns were similar in that HSP70 was more stable in the resistant cultivars throughout the application when abiotic stresses were applied to the SA resistant and susceptible tomato cultivars as compared to Israel resistant and susceptible breeding lines. A relation between infection severity and the pattern of HSP expression was found. A higher level of HSP 70 in resistant tomato plants could contribute to a lower symptom severity phenotype.

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Vita. / Digitized by Kansas Correctional Industries

Arceuthobium americanum is a flowering plant which parasitizes Pinus contorta (Lodgepole pine). This study examined branch performance of P. contorta infected to varying degrees with A. americanum.

Boschniakia hookeri Walpers has been found to parasitize the roots of Gaultheria shallon Pursh, Arctostaphylos uva-ursi (l.) Spreng, and Vaccinium ovatum Pursh, all members of the Ericaceae. This is the first documented report of the parasitism of Vaccinium ovatum. The subterranean shoot of B. hookeri originates endogenously from the perennial tubercle of the parasite. The shoot has an eustele, composed of collateral vascular bundles, arranged in four composite fronts and separated by four wide 'leaf gaps.' Pits on metaHylem vessels range from nearly circular to laterally elongated. Rll vessel members have simple perforations. Ouerall, the Hylem of subterranean shoots contained relatively few tracheary elements. Phloem is abundant, with radial series of sieve tube members that have simple sieve plates. Brachysclereids forming large clusters are positioned to both the inside and outside of the vascular bundles. Abundant stomata are present and frequently raised above the shoot surface. RHillary buds along subterranean shoots are capable of producing flowers or branch shoots. Considerable secondary growth occurs in maintained shoots below developing branch shoots. This manifested by the additional production of secondary Hylem and phloem, and the formation of a protective cuticular epithelium. This study of B. hookeri supports the view that the short and fleshy character of the subterranean shoot, increased presence of phloem, reduction of leaves to scales, and a perennial nature are specializations associated with the holoparasitic mode of life.

No description available.