Dissertations / Theses on the topic 'Seedling pathogens'

The Janzen-Connell mechanism is proposed to maintain plant diversity: predators and diseases of seeds reduce the number of seeds that survive near a parent tree, but allow seeds far from the parent tree to grow into adulthood. In the area where seeds don’t survive, seedlings from other tree species which are not affected by the seed consumer can grow. At large scales, this effect is thought to increase overall plant diversity. Soil-borne pathogens can contribute to seed mortality in this way, but we don’t know how important different parts of their lifecycle are in creating Janzen-Connell patterns. To determine the role of soil-borne pathogens in the development of Janzen-Connell patterns, we constructed a simulation model to examine how tree and pathogen characteristics affect plant spatial patterns. Under specific combinations of tree and pathogen characteristics, we found that pathogens could create Janzen-Connell patterns. The most important parameters were how far trees dispersed their seeds, and how many seeds a tree produced in a year. These characteristics determined how much of an impact the pathogens were able to have on the tree population because they rely on the density of seeds to determine how far they can spread and how many spores they will produce.

Seedling diseases are both common and destructive on soybean, Glycine max (L.) Merr. They are caused by a variety of plant pathogens including fungi, bacteria, and oomycetes. The goal of this research was to identify fungal pathogens causing seedling diseases of soybean through a multi-state effort. Two different approaches were used for genus and species determination, one relying on morphological features using microscopy and the other through DNA sequencing of specific genetic loci using up to three different barcodes. The internal transcribed spacer (ITS), the elongation factor EF-1α, and the intergenic spacer (IGS) regions were sequenced for the various isolates to confirm speciation. Data produced from the study includes identification of fungal isolates from eight states in the Unites States, each one contributing approximately 150 isolates. Data also involves correlating species and genus distributions to factors including geographical distribution, cultural practices, soil type, and climatic conditions. More than 3,000 fungal isolates were processed for identification to help determine the predominant causal agents of seedling diseases on soybean in the production fields where the isolates were collected. Our findings showed that Fusaria were the most predominant of the genera identified followed by Trichoderma species. Of sixty-one different species identified over the course of two years of the study, F. oxysporum was the most abundantly isolated. Evidence was shown that different cultural practices and environmental factors do have effects on the distribution of the various species seen in soybean fields with seedling diseases. Through the two-year efforts of seedling diseases causing fungal identification studies, further research studies were developed and pursued. In addition to the fungi believed to be contributing to seedling diseases, we also identified fungal isolates with potential to serve as biological control agents (BCAs). We found fungi, e.g. Trichoderma species, known for their control potential and proceeded with in vitro and in vivo studies to further understand their efficacy and mechanisms on a variety of pathogens with a focus on the sudden death syndrome (SDS) pathogen, Fusarium virguliforme. Species of the Trichoderma genus more significantly inhibited pathogen growth than other genera tested and T. harzianum was the most efficient of Trichoderma species.

Soil inoculum density and the incidence of black root rot of cotton caused by Thielaviopsis basicola were monitored in two adjacent fields planted mid-April to Gossypium hirsutum 'Acala 1517' at Duncan, AZ (1160 m elev.). Forty soil cores (3.5 x 15 cm) were taken from the root zone and 80 plants were collected biweekly in the two fields from 5/7/87 to 9/28/87. The inoculum density (cfu/g air-dried soil) was determined by plating soil dilutions onto a selective medium. Disease severity was rated on a scale of 1 (slight cortical decay) to 4 (severe cortical decay). Mean inoculum density in Field 1 soil was 65 cfu/g soil and 20% of the seedlings were infected with a severity rating averaging 1.6. In Field 2 the inoculum density, percentage of infected plants, and disease rating were 225 cfu/g soil, 93, and 3.2, respectively. No cortical decay was noted after June 6 in either field. Yields were similar in both fields. Field and laboratory studies indicate that high disease incidence of black root rot caused by T. basicola results in reduced incidence of seedling disease caused by Rhizoctonia solani. For example, 1 mo after untreated seeds of G. barbadense 'Pima S-6' were planted into a cotton field in Coolidge, AZ with natural inoculum levels of 12 cfu/100 g soil and 225 cfu/g soil of R. solani and T. basicola, respectively, 98% of the plants were infected by T. basicola while only 2% were infected by R. solani. Eleven out of the 12 R. solani propagules were pathogenic to cotton. The same levels of inoculum, were added to autoclaved field soil in growth chambers at 18 C and 5200 lux light in four treatments. The first treatment, T. basicola alone, caused a 98% incidence of black root rot. Rhizoctonia solani alone resulted in a 38% incidence of disease, whereas inoculum of T. basicola and R. solani, together caused a 98% and 17% incidence of disease caused by T. basicola and R. solani, respectively. Simultaneous inoculations of 4-day-old G. hirsutum 'DP 90' seedlings on water agar plates with inoculum of T. basicola and R. solani resulted 5 days later in a significant decrease in infection by R. solani as compared to that in the seedlings inoculated with R. solani only.

[Abstract]Crown rot, caused by Fusarium pseudograminearum (Fpg), is an important soilborne disease of wheat and barley. The degree of crop damage depends on seasonal conditions. Typically, high moisture conditions early in the season encourage seedling infection from stubble residues. Moisture stress later in the season leads to the production of unfilled “whiteheads”. Current control relies on cultural practices and sowing of partially resistant varieties. In order to understand the nature of partial resistance, I have examined the patterns of disease symptom development and pathogen spread in susceptible and partially resistant tissues of both pot-grown wheat, barley and oat seedlings and field-grown inoculated wheat trials. Further research was conducted to determine whether differences in pathogenicity occur amongst a small subset of Australian Fpg isolates. Seedling experiments confirmed that differences in disease ratings between susceptible and partially resistant genotypes are detected in younger leaf sheaths of older seedlings. At later harvest times differences between these genotypes are not significant in older leaf sheaths. Re-isolation of Fpg from inoculated seedlings has shown that each tissue was infected later in partially resistant genotypes compared to susceptible ones with a significantly lower number of isolations recorded at each harvest time in 42 day old seedlings. Barley cultivars were rapidly infected by the pathogen and exhibited high levels of disease symptoms. By comparison levels of infection in oats were low compared to all other genotypes. No significant differences between genotypes were observed in coleoptile tissues, either in fungal colonisation or development of disease symptoms. Disease development in the subcrown internode varied between lines/cultivars but was not representative of the relative susceptibility of each genotype. The pathogen did not appear to invade plant tissue via the vascular system but rather spread directly across the stem from leaf sheath to leaf sheath. Field trials were designed to study disease symptom development and localisation of Fpg hyphae in all expanded tissues (excluding head and roots) in wheat genotypes of known susceptibility to crown rot. Plants were harvested at approximately fortnightly intervals throughout the growing season. The main effects and interactions of harvest, genotype and tiller on each plant part were examined with a detailed statistical analysis of differences seen in these factors between susceptible and partially resistant wheat genotypes, in two inoculated field trials. While differences between genotypes were mostly not significant at each harvest when disease rating or isolations from leaf sheath tissues were examined, important differences between susceptible and resistant genotypes were seen in disease developments and Fpg infections of stem tissue in field trials. Restriction of pathogen growth and symptom development was more pronounced in the tissues of 2-49 (possesses seedling resistance) than in the field resistant Sunco. At present, the mechanisms that lead to these resistance responses are unknown. The pathogenicity study aimed to determine whether 7 Fpg isolates and a mixed inoculum differed in ability to cause crown rot in 9 wheat genotypes ranging in susceptibility to this disease. Although a genotype*inoculum interaction was significant, there is no evidence of stable pathogenic races in the isolates examined in these experiments. The growth of all isolates was partially inhibited in a consistent manner on resistant genotypes when compared to very susceptible genotypes. These results confirm significant differences in the aggressiveness of Fpg isolates on wheat, evidenced by variation in mean disease severity between isolates growing on a range of host genotypes.

Soil borne fungi isolated from forest areas and nurseries in North east of Scotland using baiting techniques, were identified using classical taxonomy and molecular methods (PCR amplification of ITS regions; restriction digestion; sequencing of PCR products) as Fusarium lateritium, F. tricinctum, F. sambucinum, Phytophthora cinnamomi, Pythium ultimum var. ultimum and Rhizoctonia binucleate (Ceratobasidium sp.). Virulence was tested in vitro on young seedlings of Pinus sylvestris and Alnus glutinosa, and Koch's postulates fulfilled through reisolation of the pathogens and confirmation of fungal penetration into host tissues. Root growth was measured using the Winrhizo program, and dry weights recorded. Symptoms on aerial parts were assessed using a categorical scale from 0 (healthy) to 5 (damage > 76%). Fusarium spp. caused significant different (P 0.01) symptom intensity on both host plants. However, no significant difference in root growth was found between treatments and control (P 0.05). The effects of different compost treatments on disease development in seedlings of both hosts inoculated with the same fine root pathogens was tested in the glasshouse confirming the virulence of the fungal pathogens on P. sylvestris and A. glutinosa seedlings. Although mean dry weights of P. sylvestris and A. glutinosa varied between compost treatments, differences were not significantly different. Isolation, characterization and identification of bacterial isolates, Bacillus subtilis B1, fluorescent pseudomonads B4 and B5 with antagonistic action against pathogens were also carried out. These isolates along with the known bacterial antagonists Bacillus subtilis MB600, MB205 and Pseudomonas corrugata R117 were used for biological control in vitro and in planta experiments using Alnus glutinosa or Pinus sylvestris seedlings. All bacterial isolates colonized root systems of both tree species. Higher numbers of bacterial cells were observed on roots of A. glutinosa than on P. sylvestris roots. High bacterial cell numbers were observed in plants of both tree species inoculated with fluorescent pseudomonads B4 or B5. In vitro antagonism on agar plates, indicated by inhibition in fungal colony diameter growth, was recorded for F. tricinctum, F. lateritium and F. sambucinum, Pythium ultimum var. ultimum and Phythophthora cinnamomi with all bacterial isolates tested (P 0.05). Biological control of the fine root pathogens on Pinus sylvestris and Alnus glutinosa seedlings by bacteria semi in vivo in test tubes was carried out with various responses in both tree hosts. All bacterial treatments resulted in a lower sporangium germination rate for P. ultimum var. ultimum than was found in controls (P 0.05). Effect of the bacterial isolates separately on growth and disease development in Pinus sylvestris and Alnus glutinosa seedlings inoculated with the pathogens under glasshouse conditions using autoclaved compost was tested. The bacterial isolates had various effects against the pathogens, although in most cases no significant differences were observed relative to controls. Further soil-based trials were carried out in the glasshouse to achieve control of root disease development on Pinus sylvestris and Alnus glutinosa using a combination of different antagonists, based on a mixture of the bacterial isolates used previously and Trichoderma koningii (TC6-Colombia). None of the antagonistic treatments showed a clear antagonistic effect in Pinus sylvestris against the fungal infections compared to control plants inoculated with the pathogens alone. In contrast, in Alnus glutinosa plants T. koningii co-inoculation improved plant growth in several of the growth parameter measured.

Lettuce (Lactuca sativa L.) seedlings diseases caused by soilborne pathogens are characterised by root rot, stem rot and damping-off of the seedlings that can occur at any time during growth. Fusarium solani, Pythium ultimum and Rhizoctonia solani are known to be the important destructive pathogens of lettuce, causing severe yield losses in South Africa. The aim of this research was to evaluate the effects of three selected fungicides to control these pathogens on lettuce seedlings. In this study the fungicides metalaxyl (Apron®), fludioxonil (Celest®) and mefenoxam (Subdue®) were applied at two concentrations as single and double doses on lettuce seedlings to determine their efficacy to control the pathogens Fusarium solani, Pythium ultimum and Rhizoctonia solani after significant reduction of mycelia growth was observed in vitro. Cultures of P. ultimum (UPGH024), R. solani (UPGH122) and F. solani (UPGH122) were obtained from the culture collection of the Department of Microbiology and Plant Pathology, University of Pretoria and cultivated on PDA for 2 days at 25ºC. Pasteurised soil was artificially inoculated with these pathogens. For the first experiment lettuce seeds were planted in polystyrene seedling trays at a depth of 1.0 cm. There were four replications of 50 seeds per treatment. In Experiment 2 pots (12 cm x 7 cm) were filled with pasteurised growing medium and 3-week old seedlings were transplanted. There were three replications of six pots containing three plants each. Seedling trays and pots were drenched with fungicides and placed in a randomised block design in a controlled environment room at 20- 26°C with a 12h-light/dark regime. The seedling trays and pots were rotated daily in the room. Seedling trays and pots were watered daily to maintain field capacity. The seedlings were able to grow larger in the pots than in seedling trays. It was confirmed that the treatment with fludioxonil (Celest®) at double and single dose inhibited the growth of the three fungi F. solani, P. ultimum and R. solani on lettuce seedlings without causing phytotoxicity. All three fungicides significantly reduced the diseases caused by the three pathogens. These findings are consistent with previous reports that fludioxonil, metalaxyl and mefenoxam can control oomycete fungi. There are few registered fungicides for the control of Fusarium solani, Pythium ultimum and Rhizoctonia solani on lettuce, therefore further work will aim to confirm these results in the field.<br>Dissertation (MInstAgrar)--University of Pretoria, 2008.<br>Microbiology and Plant Pathology<br>unrestricted

The defence mechanisms expressed in roots of Pinus sylvestris seedlings challenged with fungal pathogens were investigated, and a comparison was made between the expression of defences in non-mycorrhizal and mycorrhizal seedlings. Papillae were formed by cortical cells of non-mycorrhizal seedlings infected with Cylindrocarpon destructans. Histochemical evidence was obtained for pectic materials comprising an important polysaccharide component of these structures, and for the deposition of polyphenolic compounds also. Proton induced X-ray emission (PIXE) microanalysis indicated that insoluble calcium levels were elevated in papillae relative to normal cell walls. Although papillae appeared important in protecting cortical cells against penetration by fungal hyphae, a primary role for the wall appositions in the resistance of seedlings of Scots pine against root pathogens could not be proven. Although phytoalexins were not detected in the roots of Scots pine seedlings following infection with C. destructans, the mean content of an abietic acid fraction (comprising six compounds, of which only dehydroabietic acid could be positively identified), increased from 5.2 to 9.7mg g^-1 dry weight. This fraction exhibited some antifungal activity. -related proteins induced de novo by infection could not be detected, but several constitutive apoplastic proteins, including some with chitinase activity, appeared to increase in the needles of root-infected seedlings. The formation of ectomycorrhizae with Pisolithus tinctorius, Suillus bovinus and Hebeloma crustuliniforme did not itself induce papilla formation in the roots of P. sylvestris. Evidence was obtained to suggest that the response was suppressed when mycorrhizal seedlings were challenged with C. destructans. Results highly suggestive of the induction of systemic resistance in P. sylvestris seedlings, consequent upon mycorrhizal infection, were obtained. In seedlings grown in vitro the survival rate of mycorrhizal seedlings challenged aerially with Botrytis cinerea was 37.5% compared with 7.1 in seedlings grown gnotobiotically. However, the physiological mechanisms by which this protection was imparted remain to be determined.

In this study, we have demonstrated that inoculation of bean seeds with non-pathogenic binucleate Rhizoctonia (np-BNR) at sowing protected bean seedlings from infection of R. solani. Using quantitative real-time RT-PCR (QRT-PCR), transcript levels of defense genes encoding 1,3-beta-glucanase (GLUC), phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS) in one-week old bean seedlings was monitored during np-BNR and R. solani interaction. The results revealed that protection effect of np-BNR correspond to a systemic suppression of these three defense genes' expression from significant higher level elicited by R. solani to the level of non-infected plants. This indicates that bio-protection by np-BNR isolates is not correlated to activation of these three defense genes' expression. Similar suppression was achieved for pre-colonization of bean seedlings with arbuscular mycorrhizal (AM) Glomus introradices on GLUC gene expression, although the AM fungus did not significantly reduce rot symptoms. Possible mechanisms implicated in down-regulation during plant-pathogen and np-BNR or AM interaction are discussed.

The processes that determine the structure of plant communities are of considerable practical and theoretical interest. Natural enemies such as herbivores, seed predators and pathogens provide one potentially important influence on plant diversity. I investigated the effects of natural enemies on plant diversity in two contrasting, species-rich plant communities (tropical forests in Panama and temperate grasslands in the UK), focusing on pre-dispersal seed predation by insects, and the mortality of seeds and seedlings caused by soil fungi. In Panama I found that pre-dispersal insect seed predators generate significant levels of mortality in multiple tropical tree species, with high heterogeneity in predation rates among individuals and at different forest sites. Insect seed predators were highly host-specific, consistent with a role in enhancing plant diversity. At Upper Seeds, a calcareous grassland site in the UK, I used manipulative experiments to show that soil fungi increase the diversity of plants propagating from soil seed banks. A parallel experiment in Panama, mimicking germination under light gap conditions, revealed differential effects of fungi among sites, with fungicide treatment appearing to increase the diversity of propagated seedlings at some sites but reducing it at others. These results suggest that the influence of soil fungi on pre-emergence mortality can alter plant diversity, even when post-emergence mortality from fungal pathogens is limited. In Panama, I also tested whether enemy-mediated mortality increases with rainfall, potentially contributing to the positive regional correlations widely observed between precipitation and plant diversity. In contrast to predictions, neither pre-dispersal insect seed predation nor the influence of soil fungi on seedling recruitment were affected significantly by site humidity, or (for soil fungi) with experimentally manipulated soil moisture levels. Overall, my results provide evidence that pre-dispersal seed predators and soil fungi can affect plant recruitment and diversity at early life stages, with potential consequences for the community structure of adult plants.

Tropical rainforests contain exceptionally high biodiversity and account for >30% of the world's carbon fixed by photosynthesis. Consequently, there are compelling reasons to deepen our understanding of the mechanisms that maintain these highly diverse forests and of the potential long-term threats to their preservation. An important process shaping tropical plant communities is negative density dependence (NDD). NDD occurs when plant performance is negatively impacted by increased neighbourhood density. Reduced performance at high neighbourhood density is thought to arise through ecological interactions between plants and their natural enemies. Thus in a healthy ecosystem, trophic interactions play vital roles as mechanisms driving NDD and are important as dispersers facilitating escape from NDD mortality. However, interruption to ecological processes caused by human activities, such as hunting, can perturb NDD interactions and cause cascading effects throughout an ecosystem. In my thesis I investigate the role of dispersal and mortality in NDD dynamics of tropical tree communities, as well as investigating local and global impacts of removing ecological interactions in tropical rainforests. In my thesis, I begin by addressing the presence and variation in strength of NDD among tree species and ontogenetic stages, the mechanisms driving NDD, and the role of trophic interactions in this process. The Janzen-Connell hypothesis predicts that host-specific natural enemies drive NDD by selectively reducing conspecific density, and increase diversity by suppressing competitive exclusion, thus allowing heterospecifics to persist. In chapters 2 and 3 of this thesis, I show that mortality driven by conspecific NDD is prevalent at the early life stages, and this effect is considerably stronger during the year after germination. Furthermore, this process is driven exclusively by host-specific fungal pathogens, which cause mortality selectively among conspecifics and drive diversity. As seedlings age beyond their first year, NDD interactions become less impacted by conspecifics but are impacted by closely related neighbours or by general neighbourhood density, representing changes in the mechanism driving NDD as seedlings age, and a decline in host-specificity of natural enemies. Equally, relative growth rates (RGR) are reduced under high neighbourhood density irrespective of species identity. Results suggest insect herbivores are the strongest driver of reduced RGR but not mortality under increased neighbourhood density. As a consequence of stronger inter than intra-specific NDD effects on RGR, insects had no impact on seedling diversity in the short term. This study supports assertions that regionally rare species experience stronger NDD than common species, accounting for the high variability in species relative abundance in the tropics. In the second part of my thesis, I address the role of large vertebrate dispersers in shaping tropical tree communities and the consequences of defaunation for tree assemblage and carbon storage. Dispersal allows seeds to escape NDD and persist to reproductive maturity and is therefore vital for the maintenance of diversity. Vertebrates disperse the seeds of more than 70% of neo-tropical tree species. However, many large vertebrates are becoming scarce due to widespread hunting. The decline of large vertebrates and their role as dispersers is predicted to alter tree community composition. Additionally, large vertebrates are responsible for the dispersal of large-seeded species, which are linked to species with high wood density. With wood density positively associated with carbon storage, there is a potential cascading influence of defaunation on global carbon storage. We investigate the consequences of declining large vertebrate mortality agents in chapter 3, and the consequences of declining large vertebrate dispersers in chapters 4 and 5. Although community composition is altered in a defaunated forest, species dispersed by extirpated fauna do not appear to drive this. In fact we find that many species thought to be heavily reliant on extirpated fauna manage to persist. Although it is thought that the simultaneous loss of seed predation from large terrestrial vertebrates may create compensatory effects, we found little support for this, with an absence of large terrestrial vertebrates driving only temporary changes to species diversity. Neither a loss of large frugivores or large-seeded species lead to declines in species with high wood density, but we detect a worrying decline in large stemmed species, which has negative implications for carbon storage. Overall, my thesis highlights the importance of NDD and trophic interactions, particularly fungal pathogens, at the early life stages in shaping tropical tree communities and in maintaining diversity. I provide evidence that the removal of trophic interactions among larger natural enemies and dispersers does not impact community assemblage in the directional manner found in previous studies. I provide evidence for the variability in response to trophic interactions among species and ontogenetic stages. I show disproportionate relative importance among natural enemies and dispersers in the maintenance of tropical tree assemblage, with implications for conservation and for assessing the consequences for tree diversity under the influence of degradation.

Thesis (MScAgric)--Stellenbosch University, 2012.<br>ENGLISH ABSTRACT: Botrytis cinerea Pers. Fr. [teleomorph Botryotinia fuckeliana (de Bary) Whetzel] causes serious losses of over 200 crops worldwide, including rooibos seedlings and pears. This pathogen is characterized by morphological, physiological and genetic diversity. The genetic diversity and population structure have not been investigated for B. cinerea populations in South Africa. Botrytis cinerea collected from rooibos seedlings and in pear orchards in the Western Cape of South Africa were investigated in the present study. The study was done with the aid of microsatellite markers, the amplification of mating type alleles MAT1-1 and MAT1-2 and determination of resistance towards various fungicides. Population dynamics was inferred and a similar picture emerged in both production systems. Botrytis cinerea annually causes severe losses of rooibos seedlings (Aspalathus linearis) in nurseries situated in the Clanwilliam region. Sampling was done in five nurseries and the cryptic species status of the isolates obtained was determined through restriction enzyme digestion of the Bc-hch gene. All but one (206 out of 207) of the isolates belonged to Group II or B. cinerea ‘sensu stricto’. Analysis of the B. cinerea Group II population, using seven microsatellite loci, was performed to assess the genetic population structure. Total gene diversity (H) was high, with a mean of 0.67. Two of the nurseries populations’ sample sizes were severely limited after clone correction, yet 100 genotypes were discerned among the 206 isolates genotyped. The percentage of maximal genotypic diversity (G) ranged between 16 and 68 for the five populations, with a total value of 17 for the 100 genotypes. One genotype, represented by 27 clones, was isolated from four nurseries. Relatively low but significant population differentiation was observed in total between nurseries (mean FST = 0.030, P = 0.001). The distribution of mating types MAT1-1 and MAT1-2 differed significantly from the ratio of 1:1 for the total population plus two of the nurseries’ populations. Three nursery populations had an equal mating type distribution. The index of association (IA) analyses suggests that the populations are asexually reproducing. Analysis of molecular variance (AMOVA) indicated that 97% of the total genetic variation is distributed within subpopulations. Fungicide resistance frequency against iprodione for 198 of the genotyped isolates displayed highly varying levels of resistance amongst the five nurseries. The mean total incidence of resistance towards iprodione was 43%, ranging from 0% to 81% for the five nurseries. Baseline sensitivity towards pyrimethanil yielded an average EC50 value of 0.096 mg/L. Botrytis cinerea isolates were collected from pear blossoms (Pyrus communis) in four orchards. Two orchards in the Ceres area and two in the Grabouw area were sampled from. A total of 181 isolates were collected from the four orchards. Incidence of blossom infection in the orchards ranged from 3% to 17%. Overall, there was a high incidence of isolates that had only the Boty transposable element (74%) compared to those harbouring both (Boty and Flipper), simultaneously (transposa, 24%). One isolate examined had the Flipper element only. Cryptic species status according to restriction enzyme digestion of the Bc-hch gene indicated that all the isolates belonged to Group II or B. cinerea ‘sensu stricto’. Analysis of the Group II population, through the use of seven microsatellite loci, was performed to assess the genetic population structure. Total gene diversity (H) was high, with a mean of 0.69 across all populations. Although two of the subpopulations displayed a high clonal proportion, overall 91 genotypes were discerned among the 181 isolates. The percentage of maximal genotypic diversity (G) ranged between 18 and 33 for the four populations, with a total value of 14 for the 91 genotypes. One genotype, represented by 27 clones, was isolated from all orchards. Moderate, but significant population differentiation was present in total among orchards (mean FST = 0.118, P = 0.001). The distribution of the mating types, MAT1-1 and MAT1-2, did not differ significantly from a 1:1 ratio for the total population as well as the subpopulations. Index of association (IA) analyses, on the other hand, suggests that the populations reproduce asexually. Analysis of molecular variance (AMOVA) indicated that 88% of the total genetic variation is distributed within subpopulations, 9% between subpopulations and only 3% between production areas. Fungicide resistance frequency against fenhexamid, iprodione and benomyl varied, with the highest levels of resistance present against benomyl and low levels of resistance seen towards iprodione and fenhexamid. In conclusion, this study has shown that there exist within the studied populations of B. cinerea, obtained from rooibos nurseries and pear orchards, an adaptive capacity to overcome current means of control. The use of population genetics to further our understanding of how plant pathogens interact and spread throughout a given environment is of cardinal importance in aiding the development of sustainable and integrated management strategies. Knowledge of the dispersal of B. cinerea in the two studied cropping systems has shed light on the inherent risk that it poses, and this together with knowledge of the levels of resistance that occurs should serve as an early warning to help divert possible loss of control in future.<br>AFRIKAANSE OPSOMMING: Botrytis cinerea Pers. Fr. [teleomorf Botryotinia fuckeliana (de Bary) Whetzel] veroorsaak ernstige verliese van meer as 200 gewasse wêreldwyd, insluitende rooibossaailinge en pere. Hierdie patogeen word deur morfologiese, fisiologiese, asook genetiese diversiteit gekenmerk. Die genetiese diversiteit en populasie-struktuur van B. cinerea populasies wat in Suid-Afrika voorkom, is nog nie ondersoek nie. Botrytis cinerea verkryg vanaf rooibossaailinge en in peerboorde in die Wes-Kaap van Suid-Afrika is ondersoek. Hierdie studie is met behulp van mikrosatellietmerkers, amplifikasie van die twee paringstipe gene (MAT1-1 en MAT1-2), asook die bepaling van weerstandsvlakke teenoor verskeie swamdoders, uitgevoer. Populasie-dinamika is afgelei en ‘n soortgelyke tendens is in beide produksie-sisteme waargeneem. Botrytis cinerea veroorsaak jaarliks ernstige verliese van rooibossaailinge (Aspalathus linearis) in kwekerye in die Clanwilliam-area. Monsters is in vyf kwekerye versamel en die kriptiese spesiestatus van die verkrygde isolate is deur restriksie-ensiemvertering van die Bc-hch geen bepaal. Almal behalwe een (206 uit 207) isolaat het aan Groep II of B. cinerea ‘sensu stricto’ behoort. Analise van die B. cinerea Groep II populasie, deur middel van sewe mikrosatellietmerkers, is uitgevoer om die genetiese populasiestruktuur te bepaal. Totale geendiversiteit (H) was hoog, met ‘n gemiddelde van 0.67. Alhoewel twee van die kwekerye se monstergrootte erg ingeperk is ná kloonverwydering, is daar nogtans 100 genotipes onder die 206 isolate wat geïsoleer is, waargeneem. Die persentasie van maksimale genotipiese diversiteit (G) het tussen 16 en 68, vir die vyf populasies, gewissel, met ‘n totaal van 17 vir die 100 genotipes. Een genotipe, verteenwoordig deur 27 klone, is uit vier kwekerye geïsoleer. Relatief lae dog noemenswaardige populasie-differensiasie is in totaal tussen kwekerye waargeneem (gem. FST = 0.030, P = 0.001). Die verspreiding van die twee paringstipes (MAT1-1 en MAT1-2) het beduidend verskil van ‘n 1:1 verhouding vir die totale populasie, asook twee van die kwekerye se populasies. Die drie oorblywende kwekerye se populasies het egter ‘n gelyke verdeling van die twee paringstipes getoon. Die indeks van assosiasie (IA) analises toon dat die populasies ongeslagtelik voortplant. Analise van molekulêre variasie (AMOVA) het aangedui dat 97% van die totale genetiese variasie binne die subpopulasies versprei is. Hoogs variërende vlakke van weerstand tussen die vyf kwekerye teenoor die swamdoder iprodioon, is vir die 198 isolate wat getoets is, gevind. Die totale gemiddelde frekwensie van weerstand teenoor iprodioon was 43%, wat tussen 0% en 81% vir die vyf kwekerye gevarieer het. Fondasie-vlak-sensitiwiteit vir pyrimethanil het ‘n gemiddelde EC50 waarde van 0.096 mg/L opgelewer. Botrytis cinerea isolate is ook vanuit peerbloeisels (Pyrus communis L.) vanuit vier boorde versamel, twee uit elk van die Ceres- en Grabouw-areas. In totaal is 181 isolate vanuit die vier boorde versamel. Die frekwensie van bloeiselinfeksie het tussen 3% en 17% gewissel. Oor die algemeen was daar ‘n hoë frekwensie van isolate wat slegs die Boty transponeerbare element teenwoordig gehad het (74%) in vergelyking met dié wat tegelykertyd beide (Boty en Flipper) teenwoordig gehad het. Een isolaat het slegs die Flipper element gehad. Bepaling van die kriptiese spesiestatus met behulp van restriksie-ensiemvertering van die Bc-hch geen het aangedui dat alle versamelde isolate tot Groep II of B. cinerea ‘sensu stricto’ behoort het. Analise van die Groep II populasie, deur middel van sewe mikrosatellietmerkers, is uitgevoer om genetiese populasie-struktuur te bepaal. Totale geendiversiteit (H) was hoog, met ‘n gemiddelde van 0.69 oor alle populasies. Alhoewel twee subpopulasies ‘n hoë klonale fraksie getoon het, is 91 genotipes tussen die 181 isolate wat verkry is, onderskei. Die persentasie van maksimale genotipiese diversiteit (G) het tussen 18 en 33 vir die vier populasies gewissel, met ‘n totale waarde van 14 vir die 91 genotipes. Een genotipe, verteenwoordig deur 27 klone, was in al vier boorde teenwoordig. Gematigde dog beduidende populasie differensiasie was in totaal tussen boorde teenwoordig (gem. FST = 0.118, P = 0.001). Die verspreiding van die paringstipes (MAT1-1 en MAT1-2) het nie betekenisvol van ‘n 1:1 verhouding vir die totale populasie, insluitende die subpopulasies, verskil nie. Indeks van assosiasie (IA) analises het egter aangedui dat die populasies ongeslagtelik voortplant. Analise van molekulêre variasie (AMOVA) het aangedui dat 88% van die totale genetiese variasie in subpopulasies te vinde was, 9% tussen subpopulasies en slegs 3% tussen produksie-areas. Frekwensie van swamdoder weerstandbiedendheid vir fenhexamid, iprodioon en benomyl het gewissel, met die hoogste vlakke teenoor benomyl waargeneem, maar baie lae vlakke teenoor fenhexamid en iprodioon. Samevattend het hierdie studie getoon dat die populasies van B. cinerea wat in hierdie twee produksie-sisteme, op rooibossaailinge en in peer boorde, ondersoek is, ‘n aanpasbaarheid toon om huidige metodes van beheer te oorkom. Die gebruik van populasiegenetika as ‘n hulpmiddel om ons kennis van patogeen-interaksies en -verspreiding te verbreed, is van kardinale belang in die ontwikkeling van geïntegreerde en volhoubare beheermaatreëls. Kennis van die verspreiding van B. cinerea in die bestudeerde gewasproduksiestelsels, werp lig op die inherente risiko wat dié patogeen inhou. Dít, tesame met kennis van die weerstandsvlakke wat voorkom, kan as ‘n vroegtydige waarskuwing dien ten einde moontlike verlies van beheer in die toekoms te help teenwerk.

<p>Fungal pathogens likely play an important role in regulating populations of tree seedlings and preserving forest diversity, due to their ubiquitous presence and differential effects on survival. Host-specific mortality from natural enemies is one of the most widely tested hypotheses in community ecology to explain the high biodiversity of forests. The effects of fungal pathogens on seedling survival are usually discussed under the framework of the Janzen-Connell (JC) hypothesis, which posits that seedlings are more likely to survive when dispersed far from the parent tree or at low densities due to pressure from host-specific pathogens (Janzen 1970, Connell 1971). One of the key challenges to assessing the importance of JC effects has been to identify and quantify the effects of the large numbers of potential pathogens required to maintain host diversity. The primary objectives of this research were to (1) characterize the fungi associated with seedling disease and mortality for a number of important southeastern US forest tree species; and (2) determine if these associations are consistent with the Janzen-Connell hypothesis in terms of differential effects on seedling survival.<br></p><p>Culture-based methods and ribosomal DNA (rDNA) sequencing were used to characterize the fungal community in recently dead and live seedlings of thirteen common tree species in a temperate mixed hardwood forest (North Carolina, USA), with the goal of identifying putative seedling pathogens. Cultures were initially classified and grouped into 130 operational taxonomic units (OTUs) using 96% internal transcribed spacer (ITS) sequence similarity; 46% of all OTUs were found only once. Using rarefaction, it was concluded that the richness of the system was not fully sampled and likely included over 200 taxa (based on non-parametric richness estimators). Species richness did not differ between sampling sites or among the five most common hosts sampled. The large ribosomal subunit (LSU) region of rDNA was then sequenced for representative samples of common OTUs and refined identifications using a constrained maximum likelihood phylogenetic analysis. Phylogenetic placement verified strong BLAST classifications, and allowed for placement of unknown taxa to the order level, with many of these unknowns placed in the Leotiomycetes and Xylariales (Sordariomycetes).<br> </p><p>Next, a hierarchical Bayesian model was developed to predict the effects of multiple putative fungal pathogens on individual seedling survival, without forcing the effects of multiple fungi to be additive. The process of disease was partitioned into a chain of events including incidence, infection, detection, and survival, and conditional probabilities were used to quantify each component individually, but in the context of one another. The use of this modeling approach was illustrated by examining the effects of two putative fungal pathogens, <italics>Colletotrichum acutatum</italics> and <italics>Cylindrocarpon</italics> sp. A, an undescribed species of <italics>Cylindrocarpon</italics>, on the survival of five seedling hosts in both a maximum likelihood and Bayesian framework.<br> </p><p>Finally, the model was used to assess the impacts of these fungi on seedling survival, alone and in combination, using data on five potential fungal pathogens and five hosts. Multi-host fungi had differential effects on seedling survival depending on host identity, and multiple infections may impact survival even when single infections do not. Evaluating these interactions among multiple plant and fungal species generates a set of targeted hypotheses of specific plant-fungal combinations that could help us better understand pathogen-driven diversity maintenance at larger scales than previously possible. Building on these results, some recommendations are provided as to how the Janzen-Connell hypothesis can be re-evaluated with respect to host specificity, pathogen distribution, and environmental context.</p><br>Dissertation

Seedling blight of maize has significantly influenced field crop stands and seedling vigour over various localities and seasons. The extent of the problem is influenced by a number of factors which includes soil temperature (generally below 13 °C), waterlogged soils, inadequate fertilization, herbicide damage and fungal pathogens. The fungi generally causing seedling damping off are often involved in a complex and succession over time varying in importance depending on the field circumstances at a given time. These generally include the Pythium spp., Rhizoctonia spp. and various Fusarium spp. These have been recorded in a number of studies conducted by local researchers in the late 1980’s and early 1990’s on sorghum but to a lesser degree on maize. Uncertainty regarding the status of the etiology of maize seedling blights as maize production practices have changed dramatically in the last 10 years with increased plant populations, reduced tillage, increased crop rotation options and new short season maize hybrids. It is therefore essential to determine the present status of seedling blights in South Africa to confirm the necessity of fungicide seed treatments to ensure adequate plant densities and seedling vigour. Cob and tassel smut caused by Sphacelotheca reiliana is a disease of maize that was a problem in the 1970’s. Due to improved fertilisation, fungicide seed treatments and hybrid resistance this disease was reduced to such levels that the disease was only found to occur on research farms where seedlings were inoculated. Since 2007, the disease was reported to reach epidemic proportions on the heavy clay soils in the Standerton area. This disease has since spread over the last seven seasons to a range including northern KwaZulu/Natal, namely as far as Underberg/Swartberg, the Witbank, Ermelo, Middelburg and Delmas area in Mpumalanga and to Harrismith in the eastern Free State maize production area. This may be due to susceptible hybrids coming onto the local market or the inability of traditional fungicide seed treatments to contain infection. New and unregistered seed treatments available will be tested for their ability to control cob and tassel smut in two fields over two seasons. The aims of this dissertation were to determine the extent of the seedling blight problem in commercial fields throughout the maize industry. To determine the efficacy of fungicide seed treatments for the control of maize seedling blights using both field and greenhouse studies, and to determine the efficacy of fungicide seed treatments for the control of cob and tassel smut of maize in field trials. A total of 101 localities were sampled throughout the maize producing region of South Africa with root discolouration varying from 0 to 90 % root discolouration. Seventy different fungal species were isolated from the maize seedlings roots which include species such as Aspergillus, Clonostachus, Fusarium, Trichoderma and Penicillium. The most commonly isolated fungi which included Aspergillus niger, Fusarium solani, Fusarium verticillioides and Fusarium oxysporum were evaluated in glasshouse studies to determine their pathogenicity. Pathogenicity differed between isolates of the same fungal species, which were collected from different geographical regions, in the glasshouse studies. Field trials for seedling blight disease showed significant differences between the localities (P < 0.001) the trials were planted at, and between seed treatments. Significant season (P < 0.001) and locality (P < 0.05) differences were also found for cob and tassel smut trials planted at Potchefstroom, North-West province and Greytown, KwaZulu/Natal Province respectively. Fungicide seed treatments also showed significant differences for cob and tassel smut regarding plants infected (P < 0.001) and yield loss (P < 0.05). Overall seed treatments can be seen as an effective controlling agent for the control of seed- and soil-borne fungi on maize.<br>MSc (Environmental Sciences), North-West University, Potchefstroom Campus, 2015

Rhizoctonia solani and Pythium spp. are aggressive soil-borne fungal pathogens responsible for seed rot and seedling damping-off of many crops. With increased environmental and public concern over the use of chemicals, biological control of these diseases has been attracting more attention. However, success with this strategy depends on the development of effective antagonists, which requires repeated in vitro and in vivo tests. Bacillus spp. were isolated from a soil sample obtained from a field where sorghum and tef had been grown for at least two years. Potential Bacillus isolates were screened for their ability to inhibit in vitro growth of R. solani and Pythium sp. Among 80 isolates tested, endospore forming Bacillus spp. H44 and H51 gave highest antifungal activity against the two test-pathogens in three consecutive tests. Results demonstrated that both H44 and H51 have potential as biocontrol agents against diseases caused by these two pathogenic fungi. The interaction between three isolates of Trichoderma (T. harzianum Eco-T, Trichoderma spp. SY3 and SY4) and Pythium sp. were investigated using in vitro bioassays together with environmental scanning electron microscopy (ESEM). Visual observation on the dual culture tests revealed that hyphal growth of Pythium was inhibited by these antagonists soon after contact between the two organisms within 3-4 days of incubation. The ESEM investigations showed that all three isolates of Trichoderma grew toward the pathogen, attached firmly, coiled around and penetrated the hyphae of the pathogen, leading to the collapse and disintegration of the host's cell wall. Degradation of the host cell wall was postulated as being due to the production of lytic enzymes. Based on these observations, antibiosis (only by Eco-T) and mycoparasitism (by all three isolates) were the mechanisms of action by which in vitro growth of Pythium sp. was suppressed by these Trichoderma isolates. The reduction of seedling diseases caused by R. solani and a pythium sp. were evaluated by applying the antagonists as seed coating and drenching antagonistic Bacillus spp. (B81, H44 and H51) and Trichoderma (T. harzianum Eco-T and Trichoderma spp. SY3 and SY4). On both crops, R. solani and Pythium sp. affected stand and growth of seedlings severely. With the exceptions of H51, applications all of isoltes to seeds reduced damping-off caused by R. solani in both crops. Application of Eco-T, H44 and SY3 to sorghum controlled R. solani and Pythium sp. effectively by yielding similar results to that of Previcur®. On tef, biological treatments with Eco-T and SY4 reduced seedling damping-off caused by R. solani and Pythium sp., respectively, by providing seedling results similar to the standard fungicides, Benlate® and Previcur®. Most other treatments gave substantial control of the two pathogens on tef. Overall, Bacillus sp. H44 and T harzianum Eco-T were the best biocontrol agents from their respective groups in reducing damping-off by the two pathogens. In all instances, effects of application method on performance of biocontrol agents and adhesive on emergence and growth of seedlings were not significant. A field trial was conducted at Ukulinga Research Farm at the University of Natal, Pietermaritzburg, South Africa, to determine efficacy of biological and chemical treatments on growth promotion and reduction of damping-off incited by R. solani and Pythium sp., and to evaluate the effects of a seed coating material, carboxymethyl cellulose (CMC), on seedling emergence and disease incidence. Seeds of sorghum and tef were treated with suspensions of antagonistic Bacillus H44 or T harzianum Eco-T, or sprayed with fungicides, Benlate® or Previcur®. Application of Benlate® and Previcur® during planting significantly increased the final stand and growth of sorghum seedlings. Seed treatments with both H44 and Eco-T substantially controlled damping-off caused by Pythium, resulting in greater dry weights of seedlings than the standard fungicide. However, they had negative effects when they were tested for their growth stimulation and control of R. solani. The CMC had no significant effect on germination and disease levels. These results showed that these antagonists can be used as biocontrol agents against Pythium sp. However, repeated trials and better understanding of the interactions among the antagonists, the pathogens, the crop and their environment are needed to enhance control efficiency and growth promotion of these antagonists. Some of these biocontrol agents used in this study have the potential to diseases caused by R. solani and Pythium sp. However, a thorough understanding of the host, pathogen, the antagonist and the environment and the interactions among each other is needed for successful disease control using these antagonists.<br>Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 2003.

Cephalosporium stripe of wheat (Triticum aestivum), caused by the soilborne fungus Cephalosporium gramineum, results in significant yield reductions in dryland winter wheat crops of the U.S. Pacific Northwest. The development of resistant cultivars offers the best hope for disease control. Breeding for resistance is hampered by the long trial times inherent in screening adult plants, and by cultivar x environment interactions in field tests. The principal objective of this research was to develop and test a procedure for screening wheat seedlings in controlled environments for resistance to Cephalosporium stripe. Wheat seedlings were raised hydroponically in growth chambers, and the fungus was increased in large fermentation tanks. The seedlings were inoculated at about 12 days post-germination. Disease severity was assessed approximately seven days later using a chlorophyll meter to measure the symptoms of chlorosis and striping. In three trials, five soft white cultivars from the Pacific Northwest and four hard red cultivars from the Southern Great Plains with known levels of field resistance were tested with a Pacific Northwest fungal isolate. With one exception, chlorophyll readings ordered the cultivars appropriately, with moderately resistant cultivars ranking above susceptible cultivars. Three other moderately resistant cultivars from the Pacific Northwest also appeared in one or two trials, and were ranked properly by chlorophyll level. Chlorophyll levels of uninoculated plants were assayed to determine if differences in chlorophyll content were innate in the cultivars. The chlorophyll levels of uninoculated and inoculated seedling treatments were only significantly correlated when the cultivar Madsen, which ranks high both in resistance and in chlorophyll content, was included. In adult plants, flag-leaf chlorophyll level corresponded to intensity of Cephalosporium stripe symptoms where disease was present, and was independent of known field resistance in undiseased cultivars. The seedling screening technique was used to investigate pathogenic variability in C. gramineum. In two experiments, a total of eight cultivars from the Pacific Northwest and the Southern Great Plains were tested with three fungal isolates from each region. No evidence of virulence/vertical resistance was found. There was also no significant adaptation of isolates to greater virulence on cultivars from the same region.<br>Graduation date: 1998

Human enteric pathogen Salmonella contaminates raw produce and triggers significant economic loss and illness. Under a natural environment, Salmonella resides in soil and enters the interior of plants without causing disease or eliciting symbiotic growth. Upon being consumed by humans, complex virulence mechanisms are elicited by the specific intestine conditions, such as high temperature and humidity and lead to profound infection. The lack of effective prevention and drug treatment are largely attributed to the unclear mechanistic understanding on Salmonella association with environmental media, and in vivo host and pathogen factors required for persistent infection. We have explored the potential of deploying the model plant organism Arabidopsis thaliana to tackle this fundamental yet clinically challenging question, as Arabidopsis possesses many advantages as a model system, including enriched genomic resources, powerful genetic tools, low maintenance cost and a large collection of individual gene deletion mutants. Our preliminary data demonstrated Arabidopsis seedlings under liquid culture conditions mimicking the intestine environment were infected and killed by salmonella within 2 days upon inoculation. The Arabidopsis system possesses well-developed genetic information and the resources to study host factors required for infection on very short time scales, thus complementing traditional animal genetic studies. We aim to define the pathogen factors required for this infection. By merging the fields of extremely powerful Arabidopsis genetics and bacterial genetics/genomics, we hope to provide insight into possible new paradigms for addressing salmonella-mediated food born infection.

With an increasing realization that many agrochemicals are hazardous to animals and humans, came the desire to replace these chemical agents with biological approaches that are more friendly to the environment and human health. Microorganisms play an important role in plant disease control, as naturally occurring antagonists. Microorganisms may also have beneficial effects on plant development when applied to plant roots. Research efforts worldwide have recorded successes in biological control and growth stimulation on many crops, particularly when using members of the genera Bacillus and Trichoderma. Their use on citrus rootstock could be advantageous to nurserymen and growers in reducing the incidence of seedling mortality and increasing production. To achieve these objectives, laboratory and tunnel experiments were conducted to develop effective biocontrol agents for citrus seedlings and cuttings. Nineteen 0 ut 0 f 23 Trichoderma isolates tested in vitro against Phytophthora p arasitica sp showed antagonistic activity by hyperparasitism and four out of eight Bacillus isolates resulted in antagonism by forming inhibition zones. The positive in vitro activity of Trichoderma and Bacillus isolates on Phytophthora provided motivation step for further trials in the greenhouse to evaluate their biological control activity on citrus seedlings and cuttings. A greenhouse trial was carried out to evaluate the biological control potential of 23 Trichoderma isolates (drenched at 5 x 105 spores / rnI) and two Bacillus isolates (drenched at 1 X 106 or 1 X 108 colony forming units (CFU) / rnI) to suppress Phytophthora parasitica sp. of rough lemon (Citrus jambhirini Lush.) seedlings. Five isolates ofTrichoderma (AA12, AA5, Trichoderma harzianum (AA16), SY3F and Eco-T~ were highly effective in suppressing Phytophthora root rot, with AA12 providing the best control. The Bacillus isolates also suppressed the pathogen but were not as effective as the Trichoderma isolates. This trial was used to test for growth stimulation activity by some of the biocontrol agents. To verify these results, a further trial was carried out to evaluate growth stimulation capabilities in the absence of any pathogen. Trichoderma Isolates AA13 and AA17 caused no 111 change in seedling growth, while other Trichoderma and Bacillus isolates had an inhibitory effect on the seedling growth. This trial indicated that the biocontrol activity was affected by inoculum densities, and as a result in vitro sporulation capacity was evaluated. TrichodermaIsolate AA16 was the largest spore producer, followed by Eco-T®. Spore production was lowest from Trichoderma isolates AA4 and AA12. Growth stimulation responses of Trichoderma Isolates AA4, AA16, Eco-TID and SYN6 were further studied at four different doses (1 X 103, 1 X 104, 5 X 105 or 1 X 106 spores / ml) on rough lemon and trifoliate orange seedlings. Trifoliate oranges responded positively to 1 X 104 and 5 X 105 spores / ml of Eco-TID, but rough lemon responded negatively to all dosages of the Trichoderma isolates applied. This indicates that the inoculum density responses may be host specific. Higher population density of 1 X 106 spores / ml of all tested Trichoderma isolates had a stunting effect on seedling growth of both species. Based on t he positive results 0 f individual applications of some Trichoderma and Bacillus isolates, of the biological control agents on rough lemon seedlings against Phytophthora parasitica in an earlier greenhouse trial, their combined effect in the control of the pathogen was performed. Before carrying out a greenhouse trial, activities of the isolates to be combined were evaluated in vitro. This trial showed that Trichoderma Isolates AA16 and Eco-T®were compatible. Trichoderma isolates AA16 and Eco-T®were also found to be compatible with Bacillus Isolates B77, B81 and PHP. As a result, further in vivo trials were conducted. The tunnel trials were carried out as two separate experiments: In the first experiment, a combination of two Trichoderma Isolates A A 16 and Eco-T®was conducted assayed at 5 X 105 or 1 X 106 spores / ml, on rough lemon seedling, and cuttings and trifoliate orange and sour orange seedlings. A combination of Trichoderma isolate AA16 and Eco-T®at 5 X 105 spore / ml increased significantly the new flush biomass of rough lemon cuttings compared to AA16 alone, but was not different from Eco-TID alone. The combination of AA16 and Eco-T® achieved no change of biomass of rough lemon and trifoliate orange seedlings. The combination of AA16 and Eco-TID did not increase the root biomass of sour orange compared to AA16 or Eco-r® alone. The combination of AA16 and Eco-r® at higher doses (1 x 106 spores / ml) showed significantly better suppression of Phytophthora root rot of rough lemon cuttings but did not show disease suppression in all seedling species verities tested. In a second experiment, individual and combined effects of Trichoderma isolates (drenched at 5 X 105 spores / ml) with Bacillus isolate (drenched at 1 X 106 colony forming units (CFU) / ml) for suppression of Phytophthora root rot on rough lemon and trifoliate orange seedlings was performed. The combination of Trichoderma Isolate AA16 and Bacillus Isolate B81 increased root biomass on rough lemon seedlings compared to the combination of Trichoderma AAI6 or Bacillus PHP but was not significantly different to Trichoderma AA16 alone. Bacillus PHP combined with Trichoderma AA16 or singly had no effect on rough lemon seedlings. Combining Trichoderma Eco--r® and with Bacillus B8I or PHP did not increase biomass of rough lemon seedlings compared to Trichoderma Isolate Eco--r® alone. There was no statistically significant differences in the effects of the combinations of the Trichoderma and Bacillus isolates compared to their individual applications on the biomass of trifoliate oranges. This study established the antagonistic potential of several South African isolates of Trichoderma and Bacillus as a viable alternative to agrochemicals for controlling Phytophthora parasitica. The growth stimulation capabilities of Trichoderma isolates in terms of seedling development was also demonstrated.<br>Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2005.

碩士<br>國立屏東科技大學<br>植物保護系所<br>98<br>The purpose of this research was focused on ultilization of Trichoderma antagonists alone or amended in pangolagrass compost to test the control efficiency of cabbage seedling damping-off. A total of 29 isolates of Trichoderma spp. were isolated from soils and dead materials of plant at different areas in Pingtung. With cellophane antagonism test and dual culture, the antagonistic activity of these Trichoderma isolates was tested, and 3 isolates of Trichoderma spp. were selected based on the inhibitory effect on mycelial growth of Rhizoctonia solani AG-4, pathogen of cabbage seedling damping-off. These three antagonistic Trichoderma isolates, designated TML, TSR and TCL, could also inhibit mycelial growth of Lasiodiplodia theobomae, Pestalotiopsis euginae. Cylindrocladium sp., Colletotrichum gloeosporioides, Phytophthora capsici and Sclerotium rolfsii. Dual culture on slide between Trichoderma sp. and R. solani AG-4 showed that TML, TSR and TCL isolates could degrade the mycelium of R. solani; in addition, the hypha of Trichoderma isolate TCL could penetrate into hypha of R. solani. All of three isolates were identified as species of Hypocera koningii (anamorph: Trichoderma koningii) by comparation of the sequence of ribosomal DNA. In composting process of pangolagrass, through monitory changes of the temperature, pH value, electronic conductivity, population dynamic of microognisms, microbial activity and seed germination test in extract of compost, a mature compost could be made after 35 days fermentation. On the agar medium plates made of extract of pangolagrass compost amended with various nutrients, the mycelial growth of the T. koningii isolates was increased when rice bran or wheat bran was amended; and the sporulation was also increased when rice bran, whole wheat flour, or soybean flour was amended. The spores produced from culture of compost extract had spore germination rate of 92% or more. Isolates TML, TCL and TSR could grow well on pangolagrass compost amended with wheat bran, rice bran or wheat flour, but not with chitin amendment. When 9% wheat grain which had been grown T. koningii TML isolate was amended to diseased soil that was infested with 5% R. solani culture materials, T. koningii TML could control 100% damping-off, while 5% T. koningii TSR could control damping-off completely. When R. solani infested soil was added with 10% pangolagrass compost which had been grown T. koningii TML or TSR isolate, the control efficiency of seedling damping-off was at 21 or 33% respectively, whereas with 20% compost amendment, 73 and 82% of control efficiency, respectively.

The increasing concerns about chemical pesticides that are environmentally hazardous and the continuous development of resistance by palhogens to chemical pesticides have led to this study. Many studies have shown that some Gram-negative bacteria, such as Pseudomonas flouresens, control plant diseases and promote plant growth. In this study Gram positive bacteria, Bacillus sp., were chosen because of their ability to produce endospores. Endospores can be used in stable, dry formulations. The advantage of using endospores is their ability to survive harsh conditions such as droughts and high temperatures, which give a long shelf life to the biological control agent. Bacillus isolates were recovered from the rhizosphere of 12 different crops, and were subsequently screened in vitro for their antimicrobial activity. Of 130 isolates, 87 exhibited antimicrobial activity against the test organisms: Rhizoctonia solani, Pythium sp., Phytophthora cinnamoni, Fusarium sp., and single representatives of Gram negative and Gram positive bacteria, namely, Erwinia carotovora and Staphylococcus aureus respectively. The Bacillus isolates B77, B81 and B69 inhibited all the test organisms investigated, which suggests that they produced broad spectrum antimicrobial compounds or more than one antimicrobial compound. Of the isolates that showed antimicrobial activity, 78 of them did not inhibit Trichoderma harzianum K D, which is a registered biological control agent; indicating their potential for combined application. Selected Bacillus isolates were tested for the biological control of R. solani under greenhouse conditions in wheat, cabbage, tomato, maize, and cucumber seedlings. Bacillus isolates were applied as seed treatments, and the inoculated seeds were planted in R. solani infested speedling trays. Shoot dry weight measurement of seedlings indicated that 12 out of 19 Bacillus isolates showed significantly different shoot dry weight in wheat whereas all the isolates tested in tomato and cucumber gave significantly different shoot dry weight. No significantly different shoot dry weight was obtained for maize or cabbage. Seed emergence findings indicated that none of the Bacillus isolates gave significantly different emergence percentage on wheat, cabbage, tomato, and maize but all of them showed significantly different emergence percentage on cucumber. The results indicate that both the pathogen and the biological control agents exhibited varying levels of specificity on each crop tested. The biological control potential of the best Bacillus isolates was tested on bean and maize crops in the field. Green bean and maize seeds were coated with the selected Bacillus isolates and then sown under field conditions. For each isolate, four replicate treatment plots were established, with and without a R. solani inoculum. Percentage emergence, plant survival levels to harvesting and yield of maize cobs and green beans pods were measured. For all parameters measured the positive and negative controls were not significantly different thereby rendering the results for the entire field study inconclusive. However, Bacillus isolates B77, BII, R5 and R7 improved green bean pod yield and Bacillus Isolate B8I increased maize yield, indicating their potentials as plant growth promoting rhizobacteria (PGPR).<br>Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 2003.

Various Trichoderma and Bacillus spp. have been documented as being antagonistic to a wide range of soilborne plant pathogens, as well as being plant growth stimulants. Successes in biological control and plant growth promotion research has led to the development of various Trichoderma and Bacillus products, which are available commercially. This study was conducted to evaluate the effect of six Trichoderma spp. and three Bacillus spp. and their respective combinations, for the biological control of Rhizoctonia solani damping-off of cucumber and plant growth promotion of dry bean (Phaseolus vulgaris L.). In vivo biological control and growth promotion studies were carried out under greenhouse and shadehouse conditions with the use of seed treatment as the method of application. In vitro and in vivo screening was undertaken to select the best Trichoderma isolates from 20 Trichoderma isolated from composted soil. For in vitro screening, dual culture bioassays were undertaken and assessed for antagonisms/antibiosis using the Bell test ratings and a proposed Invasive Ability rating based on a scale of 1-4 for possible mycoparasitic/hyperparasitic activity. The isolates were further screened in vivo under greenhouse conditions for antagonistic activity against R. solani damping-off of cucumber (Cucumis sativus L.) cv. Ashley seedlings. The data generated from the in vivo greenhouse screening with cucumber plants were analysed and grouped according to performance of isolates using Ward‟s Cluster Analysis based on a four cluster solution to select the best isolates in vivo. Isolates exhibiting marked mycoparasitism of R. solani (during ultrastructural studies) viz, T. atroviride SY3A and T. harzianum SYN, were found to be the best biological control agents in vivo with 62.50 and 60.06% control of R. solani damping-off of cucumber respectively. The in vitro mode of action of the commercial Trichoderma product, Eco-T®, and Bacillus B69 and B81 suggested the production of antimicrobial substances active against R. solani. In vitro interaction studies on V8 tomato juice medium showed that the Trichoderma and Bacillus isolates did not antagonise each other, indicating the possibility of using the two organisms together for biological control and plant growth promotion studies. Greenhouse studies indicated that combined inoculation of T. atroviride SYN6 and Bacillus B69 gave the greatest plant growth promotion (43.0% over the uninoculated control) of bean seedlings in terms of seedling dry biomass. This was confirmed during in vivo rhizotron studies. However, results obtained from two successive bean yield trials in the greenhouse did not correlate with the seedling trials. Moreover, no increase in protein or fat content of bean seed for selected treatments was observed. In the biological control trials with cucumber seedlings, none of the Trichoderma and Bacillus combinations was better than single inoculations of Eco-T®, T. atroviride SY3A and T. harzianum SYN. Under nutrient limiting conditions, dry bean plants treated with single and dual inoculations of Trichoderma and Bacillus isolates exhibited a greater photosynthetic efficiency that the unfertilized control plants. Bacillus B77, under nutrient limiting conditions, caused 126.0% increase in dry biomass of bean seedlings after a 35-day period. Nitrogen concentrations significantly increased in leaves of plants treated with Trichoderma-Bacillus isolates. However, no significant differences in potassium and calcium concentrations were found. Integrated control (i.e. combining chemical and biological treatments) of R. solani damping-off of cucumber seedlings proved successful. In vitro bioassays with three Rizolex® concentrations, viz., 0.01g.l-1, 0.1g.l-1 and 0.25g.l-1 indicated that the selected Trichoderma isolates were partly sensitive to these concentrations whereas the Bacillus isolates were not at all affected. In a greenhouse trial, up to 86% control was achieved by integrating 0.1g.l-1 Rizolex® with T. harzianum SYN, which was comparable to the full strength Rizolex® (1g.l-1) application. Irrespective of either a single or dual inoculations of Trichoderma and/or Bacillus isolates used, improved percentage seedling survival as achieved with the integrated system, indicating a synergistic effect. The results presented in this thesis further reinforce the concept of biological control by Trichoderma and Bacillus spp. as an alternative disease control strategy. Furthermore, this thesis forms a basis for Trichoderma-Bacillus interaction studies and proposes that the two organisms could be used together to enhance biological control and plant growth promotion.<br>Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2005.