Thesis (M. Phil.)--University of Hong Kong, 1974. / Typescript.

Colletotrichum coccodes (Wallr.) Hughes, a foliar pathogen of velvetleaf, is being developed as a bioherbicide. Formulation of living organisms for use as pest control products presents unique problems. This research has achieved the development of an adequate formulation of the pathogen by using kaolin clay or talcum powder (1:2.79 wt/wt) as the fillers to dry conidia. Formulated C. coccodes conidia stored at 4, 30C, or at room temperature in bags permeable to oxygen remained viable and able to infect velvetleaf plants at least six months in storage. Various reported germination stimulants increased germination of formulated conidia, although not significantly, whereas increasing concentrations of cutin resulted in subsequent decreases in germination and appressoria formation of fresh as well as formulated conidia. In controlled environment experiments, 14 day-old velvetleaf seedlings were severely diseased when stearic or oleic acids were added to conidia formulated in kaolin clay or talcum powder, respectively. Combinations of germination stimulants, cutinase and/or pectinase inducers did not significantly increase germination and appressoria formation of C. coccodes conidia. Germination of fresh and formulated conidia increased, although not significantly, with the addition of 1% sucrose.

Resistance responses of Abutilon theophrasti were investigated to determine defense mechanisms of the weed against Colletotrichum coccodes and to verify if some chemical suppression of the resistance mechanism could be exploited to enhance the virulence. Induced resistance in A. theophrasti has been confirmed in treatments with C. coccodes, benzothiadiazole, bentazon, and acifluorfen. Induction of peroxidase and phenylalanine ammonia lyase (PAL) activities in the leaves that did not contact with the inducing agents was observed after the localized stresses to the first leaf or the root of the plant with those agents. alpha-Amino-oxy acetic acid (AOA), 2-deoxy-D-glucose (DDG), mannose, oxalic acid, and analogues of oxalic acid and mannose were tested to enhance C. coccodes virulence. However, the compounds did not enhance C. coccodes virulence or affect A. theophrasti growth. Strong antifungal effects, poor inhibitory effects on plant defense mechanisms, or minor dependence of A. theophrasti on the defense mechanisms that the chemicals affected could be reasons. The efficacy of C. coccodes increased in the presence of 0.25 kg a.i. ha-1 bentazon more than when C. coccodes was applied alone, while the effect of glyphosate was minimal. Peroxidase activity was strongly induced by the treatment of C. coccodes and increased over time. PAL and activation of peroxidase was inhibited in the presence of bentazon, suggesting the synergy effect by bentazon is probably due to the suppression on the two defense-related enzymes. In conclusion, A. theophrasti exploits various biochemical and morphological types of defense mechanisms against C. coccodes infection. However, the activation of the defense responses can be suppressed or by-passed in an integrated weed management system.

Volatile metabolites from headspace gas of mango cultivars Tommy Atkins and Keitt, wounded and inoculated with two pathogens, Colletotrichum gloeosporioides and Lasiodiplodia theobromae or non-inoculated controls were profiled using a GC/MS to develop a technology to discriminate diseases. Several disease discriminatory compounds were identified and classified into three groups: (i) compounds unique to only one treatment; (ii) compounds common to two or more treatments but not to all; and (iii) compounds common to all treatments but with varying in their abundance. 1-pentanol and boronic acid ethyl were detected in only Lasiodiplodia-inoculated mangoes while thujol was observed only in Colletotrichum-inoculated mangoes. Models based on significant mass ions classified up to 100% of the diseases/inoculations. The disease discriminatory compounds and discriminant analysis models developed here could be used in the early detection of postharvest diseases of mango fruit, after validation under commercial conditions.

Field and growth bench experiments were performed to assess the effect of a selective fungal pathogen of Abutilon theophrasti (velvetleaf) on various aspects of intra- and interspecific competition between this vigorous agricultural weed and soybean (Glycine max). In the absence of the foliar pathogen, Colletotrichum coccodes, A. theophrasti and soybean responded differently to the presence of conspecies or to individuals of the other species. In pure stand, the deleterious effects of intraspecific competition on reproductive output were substantially greater for A. theophrasti than for soybean, especially at lower monoculture densities. In mixtures, however, A. theophrasti reproductive performance was markedly higher than at equivalent monoculture densities, particularly at the lower mixture densities. Soybean reproduction at these lower mixture densities (10 to 20 plants m$ sp{-2}$) was severely curtailed compared with reproductive output at equivalent pure stand densities. A. theophrasti reproductive output was limited more by the presence of conspecies than by the presence of soybean, whereas the opposite trend was observed for soybean. In pure stand, application of C. coccodes had limited impact on either A. theophrasti or soybean yield. However, application of the fungal pathogen in A. theophrasti monocultures caused significant (30-44%) aboveground biomass reductions within five weeks of inoculation, in two of the three years in one field study. Eight weeks following C. coccodes inoculation, A. theophrasti biomass within inoculated monoculture plots did not differ significantly from biomass within uninoculated control plots, although height hierarchies were significantly more developed. In mixtures, C. coccodes applications caused reductions in A. theophrasti growth and reproduction when provided with an adequate dew period. Alternatively, soybean yield losses within inoculated mixture plots were generally lower than for uninoculated control plots, althoug

Colletotrichum coccodes strain DAOM 183088 is considered a potential bioherbicide for velvetleaf (Abutilon theophrasti), a devastating weed in North American corn and soybeans. Risk assessment studies have created a demand for an accurate and robust method to monitor this strain, and to distinguish it from indigenous background population of microorganisms present in the field. Safe biological control management of velvetleaf also requires comprehensive understanding of the pathogenicity determinants employed by this host-specific fungus to establish infection on velvetleaf, an aspect central to a safe biocontrol strategy task. In this study, molecular markers were designed that allow strain specific identification of the bioherbicide strain of C. coccodes and its identification within complex plant and soil matrices. An assay was developed to quantify C. coccodes from deliberate release field soil samples, in which biases caused by soil-originating PCR inhibitors were monitored on a sample per sample basis. The developed external control assay allowed for the estimation of target C. coccodes DNA quantities with normalization for the presence of PCR inhibitory compounds. Kinetic growth curves of disease development were performed for C. coccodes wild-type and T20-a (genetically engineered for hypervirulence with the NEP1 (necrosis and ethylene inducing peptide) gene) strains on velvetleaf leaves over a period of 14 days after C. coccodes infection. The wild-type strain was more efficient at infecting velvetleaf than the transgenic T-20a strain, while expression of NEP1 could not be detected suggesting that the introduced gene may not be transcriptionally active in the transformed strain, a result in conflict with previous observations. Velvetleaf and C. coccodes genes specifically upregulated at 12 and 24 h after fungal infection were cloned and differentially screened by microarrays. The resulting EST collection was sequenced and assigned to putative functions. Early gene up-regulation was confirmed by QRT-PCR analysis for type 3 metal lothionein, EREB, WRKY, and bZIP transcription factors, reticuline oxidase, ascorbate peroxidase, and ACC oxidase gene candidates. In addition, type 2, type 3 metallothionein, and bZIP gene expression profiles were investigated over a period of 14 days after C. coccodes infection, and the results indicated that C. coccodes altered the expression of all three gene analyzed.

Experiments were conducted in controlled and field conditions to determine the effect of seed size, a fungal pathogen (Colletotrichum coccodes), and soybean interspecific competition on the population dynamics of Abutilon theophrasti (velvetleaf). Seed size differences among ten individual A. theophrasti plants significantly (P < 0.001) affected seed germination and dormancy. Higher seed viability (98%) was observed among seeds having a weight above 6.0 mg. The response of A. theophrasti plants that originated from two extreme seed size groups (small <7mg and large >12mg) to the pathogen, C. coccodes, did not change over generations, and the most vigorous plants produced heavier seeds regardless of the initial seed size or infection with C. coccodes. Under field conditions, the application of C. coccodes and the herbicide, bentazon, did not affect the vegetative and reproductive biomass of A. theophrasti plants when grown in monospecific stands. However, a split application of C. coccodes and bentazon significantly reduced the aboveground biomass and reproductive output of A. theophrasti plants when grown in competition with soybean. The frequency distributions of A. theophrasti plant height, aboveground biomass, and stem diameter were positively skewed (L-shaped) when competing with soybean. However, A. theophrasti plant height and stem diameters were negatively skewed (J-shaped) and the aboveground biomass was positively skewed (L-shaped) in monospecific stands. The allometric relationships of A. theophrasti aboveground biomass and stem diameter in comparison with plant height were curvilinear when grown alone and when in the presence of soybean. However, aboveground biomass and stem diameter showed a simple linear relationship on a log-log scale in both monospecific stands and in competition with soybean.

An anthracnose-inducing pathogen, Colletotrichum dematium, was studied as a bioherbicide for Epilobium angustifolium. A comparative study involving other C. dematium isolates suggests that the isolate from E. angustifolium is a forma specialis and should be designated as Colletotrichum dematium f.sp. epilobii. The most severe damage was achieved on seedlings using a conidial density of $1 times 10 sp9$ conidia m$ sp{-2}$. Virulence decreased with plant maturity. Satisfactory levels of control were limited to long dew duration ($>$18 h) and high temperature treatments. Of various adjuvants tested, significantly higher levels of control were achieved when inoculum was sprayed in a vegetable oil emulsion (25% v/v). An inoculum buffered to acidic pH levels (pH 3.0) similarly increased level of control compared with an unbuffered conidial suspension and the adjustment to more alkaline pH levels using the citrate-phosphate buffer (pH $>$ 4.0), inhibited disease expression. In host range studies, C. dematium f.sp. epilobii was restricted to the Onagraceae family. Both Camissonia bistorta and Clarkia pulchella were susceptible whereas the fungus was highly virulent on all but one E. angustifolium ecotype. Among nine commercial tree species tested for susceptibility yellow birch (Betula alleghaniensis) was susceptible to the fungus when conidia were applied in an oil emulsion. The application of inoculum in a tank mix combination with the oil emulsion and a low rate of glyphosate provided significant growth control of E. angustifolium seedlings (7-wk-old), whereas the effectiveness of this suspension significantly decreased with plant maturity. Under controlled conditions, post-emergence application of an oil-based formulation including the ground colonized substrate of another bioherbicide candidate of E. angustifolium, Alternaria sp., significantly reduced above-ground biomass when provided a 12-h dew and applied at a rate as low as $5 times 10 sp6$ conidia m$ sp{-2}$.

Thesis (M.Phil.)--University of Hong Kong, 1974. / Also available in print.

Submitted by Marco Antônio de Ramos Chagas (mchagas@ufv.br) on 2017-04-24T17:04:25Z No. of bitstreams: 1 texto completo.pdf: 484806 bytes, checksum: d576bb5eb763685bdda5cdcd830e394b (MD5) / Made available in DSpace on 2017-04-24T17:04:25Z (GMT). No. of bitstreams: 1 texto completo.pdf: 484806 bytes, checksum: d576bb5eb763685bdda5cdcd830e394b (MD5) Previous issue date: 2003-03-10 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Neste trabalho foram conduzidos experimentos em laboratório, visando estabelecer a patogenicidade de Colletotrichum sp. ao cafeeiro (Coffea arabica L.). Isolamentos de Colletotrichum sp. foram feitos a partir de material coletado em quatro estados: Minas Gerais, São Paulo, Espírito Santo e Paraná. A partir dos isolamentos foram obtidas 180 culturas monospóricas, mantidas em sílica-gel, visando à preservação. Entre as 180 culturas inoculadas, em ramos com três rosetas de frutos verdes, houve diferença na incidência observada, tanto nos ramos com ferimento como naqueles sem ferimento, dez dias após a inoculação. A incidência máxima obtida foi de 69,5%, entretanto a testemunha, atomizada com água estéril, também manifestou sintomas e abundante esporulação de Colletotrichum gloeosporioides, resultado novamente observado quando da repetição do experimento. Isso gerou dúvidas sobre a eficiência da sanitização dos ramos e frutos verdes coletados no campo. As amostras passaram por um processo de esterilização superficial, utilizado nas etapas para isolamento de organismos endofíticos. Pôde-se observar que, mesmo superficialmente estéreis, os tecidos apresentavam abundante esporulação de C. gloeosporioides após seis dias de incubação em condições assépticas. Testes de patogenicidade foram então realizados, com e sem ferimento, utilizando-se três isolados, os quais foram inoculados em plantas oriundas de cultura de tecido, com três meses de idade, não sendo observados sintomas por mais de 20 dias após a inoculação. Porém, conseguiu-se recuperar o fungo das plantas inoculadas, mesmo depois de lavadas em água corrente, álcool 70% e cloro ativo 2%. Isso mostra que o organismo penetrou nos tecidos, entretanto sem manifestar sintomas. A partir desses resultados, concluiu-se que testes de patogenicidade de C. gloeosporioides devem ser conduzidos em plantas de cultura de tecido, a fim de se assegurar a sanidade do material a ser inoculado, visto que, mesmo após esterilização superficial do tecido, o organismo pode permanecer latente. Neste trabalho, obtiveram- se indícios de que C. gloeosporioides apresenta relação endofítica nos tecidos de cafeeiro (Coffea arabica). / This work had the objective to establish the pathogenicity of Colletotrichum sp to Coffea arabica. Isolations of Colletotrichum sp. were done from material collected in the field in four States: Minas Gerais, São Paulo, Espírito Santo and Paraná. It was obtained 180 monoconidial cultures and maintained in silica-gel, for preservation. Among the 180 inoculated cultures, in branches with 3 rosettes of green fruits, there was difference in the incidence in the wounded and not wounded branches 10 days after the inoculation. The maximum incidence was of 69.5%, however check treatment atomized with sterile water, also manifested symptoms and abundant sporulation of C. gloeosporioides, the same results was observed again when the experiment was repeated. This generated doubts on the efficiency of the sanitization of the branches and green fruits collected in the field. The tissue samples went by a superficial sterilized process, used in the stages to isolate endofitic organisms. It was observed that even superficially sterile, the tissues presented abundant sporulation of C. gloeosporioides after six days of incubation in aseptic conditions. Pathogenicity tests were then accomplished, with and without wound, using three isolates in three months old coffee seedlings of tissue culture. No symptoms observed for more than 20 days after the inoculation. However, the fungus was recovered of the inoculated plants, even after they had been washed in running water, alcohol 70% and active chlorine 2%. However, C. gloeosporioides was isolate from the inoculated tissue, although no symptoms were observed. From these results it was concluded that pathogenicity tests C. gloeosporioides, should be performed in plants from tissue culture, because, even after superficial sterilization of the tissue, the organism can be in the stage latent. In this work it was obtained, indications that C. gloeosporioides presents endofitic relationship in the tissues of Coffea arabica. / Dissertação importada do Alexandria