Bibliography: leaves 169-185 / viii, 185 leaves, [1] leaf of plates : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Plant Pathology, 1987

Thesis (M. Sc.)--University of Stellenbosch, 2002. / Includes bibliographical references. Also available via the Internet.

Thesis (doctoral)--University of Göttingen, 2002. / Includes bibliographical references (p. [102]-118).

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Eucalyptus, pine and wattle are the predominant exotic wood species used in the production of dissolving pulp in South Africa. On entering the mill, wood is chipped and stored in outdoor piles where it becomes vulnerable to microbial degradation and spontaneous combustion. Major losses of stored chips are due to high temperatures and combustion caused by heat energy released by microbial fermentation. Changes in the chemistry of the wood chips caused by the metabolic activity of indigenous microflora combined with the inherent chemical characteristics of each wood species could have a potential impact on final pulp quality and yield. Therefore the objective of this study was to analyse the microbial (bacteria and fungi) communities present in commercial wood chip piles and correlate this with changes in the chemistry of the wood in summer and winter. The molecular fingerprinting technique of Denaturing Gradient Gel Electrophoresis (DGGE) was optimized for the detection of microbial diversity in commercial wood chips. Wood chips were collected from an industrial wood yard and milled to different specifications. A total of four primer sets with GC-clamps were tested in nested PCR for DGGE analysis. 16S and 18S rRNA genes were amplified using 338f-GC/518r; 933F-GC/1387R (bacteria) and NS26/518R-GC; EF4F/518R-GC (fungi), respectively. Several gel gradients were examined to determine optimal separation of bacterial (40/60%, 35/50%, 30/60%) and fungal (35/50%, 20/45%, 25/50%) PCR-DGGE products. Comparison of the DGGE profiles revealed greater diversity in the milled wood chips amplified using primer sets; 338F-GC/518R (16S) and NS26/518R-GC (18S) with gradients of 30/60% (16S) and 25/50% (18S). Once optimized, this standardized protocol was tested against five samples to assess its applicability to woodyard samples. 16S and 18S DGGE profiles were generated and amplicons excised from gels, re-amplified, sequenced and the microorganism from which the DNA originated was determined. In the second phase a cross-sectional study of wood chip piles from a commercial dissolving pulp mill was conducted with sample collected in summer and winter using the optimized PCR-DGGE technique. Microbial strains were identified after sequencing of 16S and 18S rRNA amplicons separated by DGGE. Chemical characteristics of the wood chips were evaluated by conducting extractive analyses using HPLC. Due to unpredictable combinations of different wood species in commercial wood chip piles, the third phase involved the investigation of individual Eucalyptus species. The microflora indigenous to the two Eucalyptus species (E. dunnii and E. nitens) and a combination of the two were subjected to winter and summer simulations for one month during which samples were tested for wood chemistry properties, microflora and the final samples were used to generate dissolving pulp. Using the PCR-DGGE method eighteen bacterial and twelve fungal species were identified from the five samples collected from the commercial wood chip pile, compared to the ten bacterial and nine fungal isolates which were identified using the culturing technique and standard 16S and 18S rRNA gene sequence analysis. Predominant genera in the optimization phase of this study were Klebsiella spp. (×3), Bacillus spp. (×2), Pantoea spp. (×2), Pseudomonas spp. (×2) and Paecilomyces spp. (×2). Application of the optimized DGGE technique to samples collected from the commercial pulping mill in summer and winter revealed variable profiles indicating a range of bacterial and fungal strains that varied in intensity in the areas and seasons sampled. Seventy nine (45 in summer and 34 in winter) and 29 (20 in summer and 9 in winter) distinct amplicons representing bacteria and fungi, respectively, were visualized. Predominant genera in summer were Pantoea rodasii, Inquilinus limosus, Streptococcus sp., Klebsiella spp., Diversispora sp., Boletaceae sp., Scutellospora sp., and Ophiostoma bicolour. In winter the prevailing genera were Leuconostoc palmae, Streptococcus sp., Bacillus spp., Diversispora sp., Boletaceae sp., and Bullera sp. Lower cellulose levels in summer correlated significantly with high microbial loads and the predominance of Bacillus spp., suggesting that in warm humid environments storage should not exceed 1-2 weeks. No correlations were determined between the decreased hot water levels in winter and microbial activity, however they were correlated to increased exposure of those samples to environmental factors. Chemistry data on the wood chips imparts the quality of the wood which only permitted projection of final pulp quality. This inadequacy was addressed in the third phase which included identification of microbial strains, originating from the individual Eucalyptus species, after sequencing of 16S and 18S rRNA amplicons separated by DGGE. Fungal and bacterial species were also isolated, cultured, identified and screened for lignocellulolytic enzyme activity. Ninety two and 88% of the fungi isolated were capable of producing cellulase and xylanase, respectively. Significant correlations exist between the microflora, seasons (greater diversity and loading in summer) and the chemical and physical properties of wood chips (lower cellulose and viscosity in summer) as well as Eucalyptus species (significantly higher cellulose and viscosity for the combination and E. nitens). Indigenous microflora of each wood species may be one of the contributing factors to poor/good pulp quality, as significant correlations were made between enzyme production of microorganisms and wood chemistry which ultimately has an impact on the final pulp quality and yields. This investigation provides proof of concept that combining wood species with different deterioration rates results in an overall improvement in pulp quality and thus paves the way for a practical and applicable approach to managing quality of chips. / Thesis (M.Sc.)-University of KwaZulu-Natal, Westville, 2013.

This research has focused on the determination of natural populations in the fields, the effect of different inoculum densities on lettuce growth and a study of the association of this fungus with two nematodes (Pratylenchus penetrans Cobb and Meliodogyne hapla Chitwood). Under conditions of artificial infestation of soil the results were satisfactory, but in trials with naturally infested soil the fungus could not be detected. The effect of different inoculum densities was measured at different stages of growth, and only in those plants inoculated 2 weeks after seeding were differences significant and consistent. Some evidence of the detrimental effect of wounding the root system prior to attack by the fungus led to studies of the relationship between this fungus with either P. penetrans or M. hapla. In the first case a negative interaction seemed to exist; no significant increase of the damage caused to the lettuce was observed. In contrast, when the root-knot nematodes and P. tracheiphilum were combined there was a marked reduction of lettuce growth. The interaction was found to be additive.

Control of seed-associated micro-organisms is vital in reducing losses of plants of economic importance. Recalcitrant seeds being metabolically active and able to be stored only under conditions of high relative humidity makes it more difficult to control contaminants. Nevertheless, means need to be developed to eliminate, or at least curtail, seed-associated fungi and bacteria. The use of biological control is a highly recommended alternative to chemical control for reducing the risk of killing beneficial organisms, as well as in terms of health and environmental hazards. Furthermore, when working with seed-derived tissues, it is extremely important to optimise a method or methods to control contamination without compromising the viability or further development of the explants. The original aim of the present study was to determine whether the biocontrol agents, EcoT® and Eco77® (commercial products of the spores of Trichoderma harzianum) would effectively control/eliminate micro-organisms from the embryonic axes of Trichilia dregeana, while promoting growth under in vitro conditions. Other means were also tested for their efficacy in controlling contaminants; these were application of Benlate®, Nipastat® (a mixture of parabens), anodic water (the anodic fraction of an electrolysed dilute solution of calcium and magnesium chloride), sodium dichloro-isocyanurate (Medi-Chlor®[NaDCC]) and alginate gel encapsulation of the embryonic axes. Prior to the experiments, fungal contaminants from the embryonic axes were isolated on potato dextrose medium and identified using light microscopy. EcoT and Eco77 were initially individually tested by co-culture as conidial suspensions with the embryonic axes. A further approach used liquid culture (potato dextrose broth) as well as solid culture medium (based on sugarcane bagasse) in/on which the strains of Trichoderma harzianum had been grown. This was aimed at testing for the possible presence of compounds released by T. harzianum into the media, which might prove to be effective in curtailing/eliminating the axis-associated microflora. Among the different treatments tested, the best method was utilised to decontaminate the embryonic axes prior to minimal-growth storage (hydrated axes encapsulated in alginate gel ‘beads’). Penicillium spp. were predominant among the different fungi isolated, which included Fusarium spp., Rhizopus spp., Aspergillus niger and Aspergillus flavus. Co-culturing with T. harzianum for 24 h was successful in terms of the survival of the embryonic axes, although the roots produced were shorter than when axes were cultured alone, but had no effect in eliminating the contaminants. Longer periods of co-culturing with T. harzianum affected the germination of the embryonic axes of T. dregeana compared with axes germinated in the absence of the biocontrol agent (control). The culture filtrate negatively affected germination of the T. dregeana embryonic axes, although it was effective against the associated contaminants. Nipastat was effective in reducing the contamination, and, depending on the concentration, did not affect germination adversely. Medi-Chlor was highly effective in eliminating all the contaminants from axes in vitro. Both these treatments were therefore used to decontaminate axes before minimal-growth storage. All the NaDCC-treated, encapsulated axes examined after 14 d hydrated storage [in Magenta boxes] and after 14-42 d in polythene bags survived; however the axes stored in aluminium foil-lined bags and Eppendorf tubes soon lost viability. The recommendation is therefore made that the decontamination treatment based on use of NaDCC (or other preparations of sodium dichloro-isocyanurate) be tested on embryonic axes of a range of recalcitrant-seeded species, and, if successful, the procedure be introduced into cryopreservation protocols. The use of NaDCC has emerged as a promising method of eliminating contaminating microflora which otherwise compromise in vitro procedures, from seed-derived explants. Furthermore, containment of decontaminated encapsulated axes in sealed polythene bags offers an apparently ideal means of temporary storage and dissemination. The results should find considerable applicability when excised embryonic axes representing the germplasm of recalcitrant seeds, are cryoconserved. / Thesis (M.Sc.)-University of KwaZulu-Natal, Durban, 2013.