Daphne is a widely dispersed genus with large variation in morphology, native habitats ,and use. Unfortunately, broader acceptance of Daphne in the ornamental trade has been limited due to Daphne Sudden Death Syndrome (DSDS), a disease that kills the plant without warning. The results of this research identified Thielaviopsis basicola (Berk. et Br.) Ferr. as the causal agent for this disease. Pure cultures of the pathogen were developed and used in a germplasm screen. To evaluate Daphne germplasm in vitro, species-specific protocols were developed that alleviated two common problems in Daphne micropropagation, browning and hyperhydricity. Optimizing the concentrations of both PGRs and charcoal was able to control these problems. Selected species were evaluated for resistance against Thielavipsis basicola in both, in vivo and in vitro, conditions. The results of both methods displayed a strong correlation and indicated significant differences among the taxa. However, there were differences in disease progression rates. Typically, the in vitro challenge produced a comparable level of disease as the in vivo challenge but in two to three weeks less time. Across both screening methods, the most resistant species evaluated were D. tangutica and D. retusa, while D. cneroum was the most susceptible. Based on ITS sequences, phylogenetic relationships among selected Daphne species were established and associated with their resistance against T basicola. The phylogeny indicated that Daphne is possibly a monophyletic group. However, placement of D. genkwa remained problematic. The analysis of ITS sequences data resulted in a parsimony consensus tree with two well-supported major clades and one Glade with less support. In general, the evolutionary tree for resistance, inferred from the phylogenetic data and the results of the screening project, indicate that resistance is a derived character and that plants recently evolved this ability.

Daphne is a widely dispersed genus with large variation in morphology, native habitats ,and use. Unfortunately, broader acceptance of Daphne in the ornamental trade has been limited due to Daphne Sudden Death Syndrome (DSDS), a disease that kills the plant without warning. The results of this research identified Thielaviopsis basicola (Berk. et Br.) Ferr. as the causal agent for this disease. Pure cultures of the pathogen were developed and used in a germplasm screen. To evaluate Daphne germplasm in vitro, species-specific protocols were developed that alleviated two common problems in Daphne micropropagation, browning and hyperhydricity. Optimizing the concentrations of both PGRs and charcoal was able to control these problems. Selected species were evaluated for resistance against Thielavipsis basicola in both, in vivo and in vitro, conditions. The results of both methods displayed a strong correlation and indicated significant differences among the taxa. However, there were differences in disease progression rates. Typically, the in vitro challenge produced a comparable level of disease as the in vivo challenge but in two to three weeks less time. Across both screening methods, the most resistant species evaluated were D. tangutica and D. retusa, while D. cneroum was the most susceptible. Based on ITS sequences, phylogenetic relationships among selected Daphne species were established and associated with their resistance against T basicola. The phylogeny indicated that Daphne is possibly a monophyletic group. However, placement of D. genkwa remained problematic. The analysis of ITS sequences data resulted in a parsimony consensus tree with two well-supported major clades and one Glade with less support. In general, the evolutionary tree for resistance, inferred from the phylogenetic data and the results of the screening project, indicate that resistance is a derived character and that plants recently evolved this ability.

Daphne is a widely dispersed genus with large variation in morphology, native habitats ,and use. Unfortunately, broader acceptance of Daphne in the ornamental trade has been limited due to Daphne Sudden Death Syndrome (DSDS), a disease that kills the plant without warning. The results of this research identified Thielaviopsis basicola (Berk. et Br.) Ferr. as the causal agent for this disease. Pure cultures of the pathogen were developed and used in a germplasm screen. To evaluate Daphne germplasm in vitro, species-specific protocols were developed that alleviated two common problems in Daphne micropropagation, browning and hyperhydricity. Optimizing the concentrations of both PGRs and charcoal was able to control these problems. Selected species were evaluated for resistance against Thielavipsis basicola in both, in vivo and in vitro, conditions. The results of both methods displayed a strong correlation and indicated significant differences among the taxa. However, there were differences in disease progression rates. Typically, the in vitro challenge produced a comparable level of disease as the in vivo challenge but in two to three weeks less time. Across both screening methods, the most resistant species evaluated were D. tangutica and D. retusa, while D. cneroum was the most susceptible. Based on ITS sequences, phylogenetic relationships among selected Daphne species were established and associated with their resistance against T basicola. The phylogeny indicated that Daphne is possibly a monophyletic group. However, placement of D. genkwa remained problematic. The analysis of ITS sequences data resulted in a parsimony consensus tree with two well-supported major clades and one Glade with less support. In general, the evolutionary tree for resistance, inferred from the phylogenetic data and the results of the screening project, indicate that resistance is a derived character and that plants recently evolved this ability. / Land and Food Systems, Faculty of / Graduate

2 pp. / Originally published: 1998 / Damping off is caused by several different fungi under different environmental conditions. The fungi include Pythium, Rhizoctonia solani, and Thielaviopsis basicola. This article discusses the symptoms, environmental conditions, diseases, prevention and control methods for the damping-off caused by fungi.

Schizolobium parahybum is native to Ecuador, South America, where plantations of this tree is being established. Development of these plantations has been unsuccessful due to a serious die-back disease. A survey on the role fungal pathogens might play in disease development were conducted by isolating possible pathogenic fungi from streaks in the xylem and from machete wounds from dying trees. The primary aim of this study was to identify the isolated fungi and to determine their pathogenicity to S. parahybum through a series of greenhouse trials. Ceratocystis fimbriata, C. moniliformis, Thielaviopsis basicola, Graphium penicillioides, Ophiostoma quercus and a Pesotum species were identified as possible pathogens of S. parahybum. Inoculation trials conducted with these fungi revealed that C. fimbriata and C. moniliformis were able to cause significant lesions on young S. parahybum trees under greenhouse conditions. These fungi were, however, not consistently isolated from diseased trees and the lesions in greenhouse trials were not consistently produced. Graphium penicillioides, O. quercus and the Pesotum species were unable to cause any notable lesions. Thielaviopsis basicola caused lesions that differed significantly from the control inoculations of S. parahybum. These results were intriguing, as T. basicola is not a known tree pathogen, but is predominantly known to cause disease of agricultural crops such as groundnuts and chicory. The ability of T. basicola to cause lesions on S. parahybum initiated the second part of the thesis that dealt with a population diversity study of this pathogen. Comparison of T. basicola isolates from Ecuador and other parts of the world with isolates from groundnut and chicory in South Africa revealed that the Ecuador isolates did not originate from S. parahybum but from the carrots that was used to isolate them. Although T. basicola from carrots were able to cause lesions on S. parahybum, we did not investigate this phenomenon further as this fell beyond the scope of this thesis. Seven polymorphic primers were developed for T. basicola using the ISSR-PCR technique. These primers were used to determine the population diversity of T. basicola from groundnuts and chicory populations in South Africa. Results showed a low diversity for both populations and suggest that T. basicola was introduced to South Africa. The markers were also used to compare isolates from the groundnut and chicory populations with T. basicola isolates from other hosts and geographical regions. This indicated that T. basicola may be native to Europe from where it possibly spread to other countries. / Dissertation (MSc)--University of Pretoria, 2007. / Microbiology and Plant Pathology / MSc / Unrestricted

Les bactéries du sol produisant des antifongiques comme le 2,4-diacétylphloroglucinol(DAPG) protègent les racines des plantes vis-à-vis des champignons phytopathogènes. Néanmoins, les conditions de fonctionnement de ces populations bactériennes dans le sol restent très mal connues. Dans certains sols, dits résistants aux maladies, ces bactéries phytoprotectrices sont présentes à des effectifs importants et leur activité est suffisante pour protéger la plante malgré la présence du pathogène. L'objectif de cette thèse a été de comprendre la relation entre la résistance des sols à la maladie de la pourriture noire des racines de tabac, et la fonction de synthèse du DAPG chez les bactéries du genre Pseudomonas. Dans la situation de référence de Morens (Suisse), les sols résistants diffèrent des sols sensibles par la présence de vermiculite, argile capable de relarguer du fer. On sait que la présence de vermiculite améliore la phytoprotection assurée par les Pseudomonas producteurs de DAPG, mais les mécanismes moléculaires sous-jacents restent inconnus. Dans un premier temps, la quantification de ces bactéries par une nouvelle méthode de PCR quantitative développée ici, a confirmé que leurs effectifs sont élevés dans les sols résistants, mais aussi dans les sols sensibles, suggérant que la résistance puise plutôt dépendre d'une plus forte expression de la fonction de synthèse du DAPG. Dans un second temps, l'étude de l'expression des gènes de synthèse du DAPG en système de sol artificiel, à l'aide de la souche rapportrice P. protegens phlA-gfp, a montré que la présence de vermiculite dans le sol se traduit par une plus forte biodisponibilité du fer pour les Pseudomonas, induisant une plus forte expression des gènes de synthèse du DAPG et la protection du tabac. En conclusion, la résistance des sols de Morens à la maladie de la pourriture noire des racines est conditionnée par plusieurs facteurs abiotiques et biotiques, dont la biodisponibilité du fer qui régule l'expression des gènes de synthèse du DAPG chez Pseudomonas / Soil bacteria producing antimicrobial compounds like 2,4-diacetylphloroglucinol (DAPG) protect plants from soil-borne phytopathogens. Nevertheless, the functioning of these bacterial populations in the soil is largely unknown. In certain soils, termed disease- suppressive soils, these bacteria are present at high numbers and their activity is sufficient to assure effective plant protection in the presence of the pathogen. The aim of this thesis was to understand the relation between soil suppressiveness towards black root rot of tobacco, and the 2,4-diacetylphloroglucinol synthesis ability of certain Pseudomonas. In Morens region (Switzerland), suppressive soils differ from conducive soil by the presence of vermiculite, an iron-releasing clay. It is known that DAPG-producing Pseudomonas provide better plant protection in the presence of vermiculite, but the molecular basis of this interaction is still unknown. First, the quantification of these bacteria, through a new real-time PCR method developed here, confirmed that high numbers of DAPG-producing Pseudomonas occur in suppressive soils, as well as in conducive ones, raising the possibility that suppressiveness depends rather on a higher expression of DAPG synthetic genes. Second, expression studies of DAPG synthetic genes using a P. protegens ph/A- gfp reporter strain and artificial soil systems, confirmed that the presence of vermiculite in the soil can translate into higher iron bioavailability for Pseudomonas, triggering higher expression of DAPG synthetic genes and effective plant protection. In conclusion, black root rot suppressiveness of Morens soils is determined by several abiotic and biotic factors, among which iron bioavailability regulating the expression of DAPG synthetic genes in plant-protecting Pseudomonas

The use of matrix assisted laser desorption ionization time-oflight mass spectrometry, Fourier transform infrared spectroscopy, and other analytical means of identifying and differentiating microorganisms hold much promise. These analytical tools have been extensively assessed for their ability to differentiate bacteria and fungi. Most of this research has been coordinated in medically relevant microorganisms, but the technology can work just as well with agriculturally important microorganisms. In this thesis, these technologies were reviewed and then subsequently studied for their ability to differentiate Aspergillus species (that devastate corn and other crops yearly with aflatoxin contamination), as well as Macrophomina phaseolina and Thielaviopsis basicola which limit yields on soybean and other crops yearly. With the use of these technologies, harmful plant pathogens could be identified and subsequently treated to improve crop yields and also help to protect our nation and state’s food supply.

Thielaviopsis basicola (Berk. and Br.) is a widespread, soil-borne, pathogenic fungus which causes a root rot on tobacco (Nicotiana tabacum L.). Presently, control is achieved primarily through the use of resistant cultivars of tobacco or chemicals. In order to develop a disease prediction program, disease-inoculum density relationships must be determined. It was the purpose of this study to develop an improved procedure for estimating the populations of T. basicola in naturally infested soil. A second objective was to determine the horizontal inoculum pattern of T. basicola in tobacco field soil, and the third objective was to determine the relationship between black root rot of tobacco and inoculum density of T. basicola in naturally infested soil. Two fields in southwestern Virginia were randomly and systematically sampled to assay the population density of T. basicola in the soil. Populations of T. basicola were isolated using a modified carrot-disc baiting technique and calculated using a colonization correction factor. Frequencies of calculated population densities of T. basicola in soil samples were compared, using a Chi-square test, to the expected frequencies indicated by the Poisson, Neyman Type A, and negative binomial distributions. Temperature-tank studies were conducted to investigate inoculum density-disease relationships. Two cultivars of tobacco were grown in soil naturally infested with T. basicola. The soil was thoroughly mixed before use to preclude inoculum clumping. The results obtained with either systematic or random sampling indicated that propagules of T. basicola are clumped or aggregated in tobacco field soil, as there was a good fit of the data to the negative binomial distribution. Also, the variance to mean ratios were significantly (P = 0.05) greater than 1.0 which is indicative of a clumped pattern. Other indexes of aggregation indicated a low to moderate degree of inoculum clumping in soil. For soil temperature-tank tests, root rot incidence was 96-100% at all natural population densities for both slightly resistant cv. Burley 21 and susceptible cv. Judy's Pride plants, and T. basicola was consistently associated with rotted roots. Low R² values were obtained in regression analyses of T. basicola inoculum density versus mean percent root rot per Burley 21 plant and per Judy's Pride plant. / Master of Science