Sclerotinia sclerotiorum is a widespread plant pathogen that produces structures known as sclerotia. When sclerotia germinate they give rise to infective hyphae, myceliogenic germination, or they produce ascocarps, carpogenic germination. Biological control has usually targeted sclerotia or ascospores. The main objectives of the research presented herein were to observe the effect of a mycoparasite and fungus gnats (Bradysia coprophila) on the survival of sclerotia in vitro and in field conditions, and to study the enzymatic activity of the mycoparasite when in contact with sclerotia damaged by fungus gnats. / In this research several mycoparasites were evaluated for their efficacy to degrade sclerotia in soil. From these tests, an isolate of Trichoderma hamatum, TMCS 3 proved to be the most effective. Larvae of fungus gnats have also been reported to feed on sclerotia. When both organisms were combined in laboratory tests, fewer sclerotia survived than when the organisms acted alone. Sclerotia recovered from this treatment contained fewer viable cells when compared to sclerotia recovered from treatments with TMCS 3 or fungus gnats alone. The results obtained from field trials showed that TMCS 3 was effective at degrading sclerotia. Unfortunately environmental conditions were not always optimal for the establishment of high populations of fungus gnats. Few larvae were observed feeding on sclerotia and no significant differences were found among treatments. / Growth of TMCS 3 was studied using different carbon sources as substrates, including sclerotia of S. sclerotiorum. Biomass obtained from this latter treatment was significantly larger than on the other carbon sources tested. Enzymatic activity was also induced by the presence of sclerotia. In many cases, sclerotial exudates from mechanically damaged sclerotia or sclerotia damaged by larval feeding showed that the concentration of amino acids, carbohydrates, and electrolytes was increased as damage to the sclerotia has increased. Exudation of protein was not different when damaged and undamaged sclerotia were compared. Exudates from sclerotia with the melanized rind completely removed by fungus gnats feeding accelerated the germination of conidia of TMCS 3. These heavily damaged sclerotia also enhanced the growth of TMCS 3 when both organisms were grown together. However enzymatic (i.e. glucanase and chitinase) activity of TMCS 3 was not increased by the damage to the sclerotia. When damaged sclerotia were buried in soil infested with TMCS 3 they were degraded faster when the medulla of sclerotia was completely uncovered by larval feeding.

Growth chamber trials were performed to investigate optimal conditions for the small scale production of isolate M12-4A of Fusarium oxysporum on substrate materials that are locally available to subsistence farmers in West Africa. Field trials were conducted in Mali to evaluate the effectiveness of F. oxysporum for the control of Striga hermonthica and to determine the host range of F. oxysporum. F. oxysporum grew and colonized substrates over a range of temperatures (24, 28 and 32 C). Chopped sorghum straw pieces, straw fibres, and glumes supported abundant mycelial growth. Full colonization of the substrates was observed within 10 days. Production of infective propagules (microconidia and macroconidia) was optimum at 28 C. Optimum wetness of the substrates was obtained by soaking straw or glumes overnight. In field studies, the incorporation of 2.6 g of dried ground straw inoculum per sorghum seed pocket (120 cm$ sp2$), at a depth of 5 or 10 cm, resulted in a 60% reduction of emerged S. hermonthica 82 days after sowing. At harvest, biomass of Striga was also reduced by 70% and sorghum grain yield was almost doubled compared with the control. Sorghum, millet, maize, rice, fonio, cotton, cowpea, groundnut, okra and sorrel were immune to isolate M12-4A.

Fusarium oxysporum M12-4A fungus is being evaluated for the biocontrol of Striga hermonthica, a parasitic weed of African cereal crops. The production of M12-4A inoculum was assessed in four Malian villages using local technology and substrates. A delivery system using arabic gum to temporarily glue inoculum powder onto the crop seed was tested. In controlled conditions, coating of sorghum seeds with arabic gum and inoculum powder did not affect seed germination or inoculum viability. However, one week at 40°C significantly decreased the viability of the inoculum by 31%. Fungus growth and chlamydospore germination were also reduced by temperatures of 34 and 36°C. M12-4A was susceptible to the fungicide thiram (ED50 = 38.5mug). Field trials were conducted in Mali to evaluate the large-scale efficacy of the seed coating technology. F. oxysporum M12-4A was detected from some S. hermonthica tissue and soil samples using specific primers and Real Time PCR.

No description available.

Bacillus thuringiensis subsp. isrealensis (B.t.i.) produces a crystalline endotoxin specific for some larvae of mosquitoes that are vectors of the malaria parasite and other infectious diseases. Fragments were obtained from the 108 kb plasmid from B.t.i. strain 4Q2 which encodes several proteins comprising the delta-endotoxin. These DNA fragments were inserted into the hybrid cyanobacterial cloning vector, pTNTV, downstream from its powerful lambda promoter, and the chimaeras were transformed into Escherichia coli. Ampicillin resistant transformants were screened with radioactively labelled oligonucleotides whose sequences were determined from the published sequences of the B.t.i. 130 kDa polypeptide. Clones showing hybridization were used in bioassays to determine their level of toxicity to the fourth instar larvae of the Aedes aegypti mosquito. Twelve clones were found that demonstrated toxicity which was statistically significantly greater than that observed in controls. Plasmid DNA from some of these clones was isolated, cut with restriction endonucleases, and viewed through agarose gel electrophoresis to confirm that B.t.i. fragments had been inserted into the vector. Future work will investigate the expression of these cloned toxin genes in transformable cyanobacteria and will determine their subsequent activity against the fourth instar larvae of Aedes aegypti and Anopheles quadrimaculatus. / Department of Biology

The purpose of this study was to examine the independent and combined effects of thermal biofeedback and thermal suggestions on peripheral temperature control. Four treatment groups were compared: biofeedback, suggestion, combined biofeedback-suggestion, and a control group receiving only instructions requesting that the subjects attempt to raise their peripheral temperature. All groups received these same response-specific instructions. It was hypothesized that the group receiving the combined treatment would demonstrate the greatest amount of peripheral temperature control and that the control group would demonstrate the least.40 subjects participated in the study (10 per group). Before attending three treatment sessions, each subject attended two baseline sessions. Temperature change was computed from the end of a stabilization period to the end of a 15 minute training period. This temperature change on baseline days represented the subject's natural drift in peripheral temperature. Baseline day changes were subtracted from training day changes in order to control for each individual's natural drift. The resulting change scores were used as the dependent variable.A 4 X 3 ANOVA with repeated measures on the last factor was the statistical design used to analyze the data. There were no significant effects found at the .05 level. The results of this study are therefore indicative of the following conclusions:1. Biofeedback, suggestion, or a combined biofeedback, suggestion method, when used in conjunction with response-specific instructions for raising peripheral temperature, are not significantly different from each other in their effects upon peripheral temperature control. Also, the effects of these treatments on peripheral temperature control are not significantly different from the effects of a treatment consisting only of response-specific instructions.2. Regardless of treatment received, peripheral temperature control is not effected by the amount of training received over three training sessions.3. The effects of the different treatments upon peripheral temperature control is not dependent upon the amount of training received over a three session period of time.Although not significantly lower, the mean of the control group consistently demonstrated the poorest level of peripheral temperature control. All four treatment groups were able to demonstrate peripheral temperature control.

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.

Late blight, caused by Septoria apiicola Speg., is the most important disease affecting celery in Quebec. Biological control was investigated as an alternative to conventional chemical control of late blight. Two hundred and four bacterial isolates were collected from celery leaves, and muck and mineral soils of celery fields in south-western Quebec. Two experiments were conducted to screen the bacteria for antagonism toward Septoria apiicola: one on agar to test for inhibition of pycnidial formation, and the other on leaf disks to test for inhibition of germination of conidia. From these two experiments, 18 isolates were selected that prevented pycnidial formation in an inhibition zone $ ge$1.0 cm wide and reduced germination to below 30% of the control. These isolates were re-evaluated for inhibition of germination on leaf disks. A bacterial suspension (10$ sp7$ cells/ml) was incubated on leaf disks for 24 hours before a suspension of S. apiicola conidia (150,000 spores/ml) was applied, and the disks were incubated for 25 hours. Four isolates reduced germination to $ le$19% of the control. These isolates were tested on plants in a greenhouse. None of the isolates was able to reduce the number of late blight lesions compared to the control.

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.

Stachybotrys elegans is a mycoparasite of the soilborne plant pathogen fungus Rhizoctonia solani. The mycoparasitic activity of S. elegans is correlated with the production of cell wall-degrading enzymes such as chitinases. This study details the cloning and characterization of the cDNA, sechi44, that encodes an extracellular endochitinase. The expression regulation of sechi44 was altered when S. elegans was in interaction with its host, R. solani, and also when the mycoparasite was grown on minimal media amended with different carbon and nitrogen sources. Direct contact with R. solani significantly upregulated sechi44 expression which followed a cyclical pattern suggesting that this gene has a role not only in mycoparasitism, but also in linear growth of the mycoparasite. The addition of high concentrations of glucose and ammonium triggered a decrease of sechi44 expression suggesting that sechi44 is subject to glucose and ammonium repression. In a separate study, several genes (1016 clones) whose transcription was substantially up-regulated during the mycoparasitic interaction were identified using SSH and microarray analysis. Twenty-five percent (261 clones) of these were sequenced and assigned to putative functions. Among them, 15 expressed sequence tags (ESTs) were identified in R. solani whose functions were related to defense while the majority of ESTs were identified in S. elegans and assigned functions related to toxin metabolism, pathogenic process, stress response., multidrug resistance, apoptosis, transport, ATP synthesis, replication, transcription and DNA repair, translation, transduction, protein degradation, and ribosomal protein. The overexpression of 13 selected genes of S. elegans was validated and confirmed using quantitative reverse transcription polymerase chain reaction (QRT-PCR). The temporal gene expression of nine genes was monitored when the mycoparasite was grown on R. solani (host) and Sclerotinia sclerotiorum (non-host) mycelia and sclerotia. Some genes such as seglu, selec, and se151 were completely inhibited by the presence of non-host hyphae suggesting that these genes play an important role during mycoparasitism. Also, the absence of these corresponding transcripts suggests that the non-host produces transcription inhibitors. As expected, gene expression of cytochrome P450 was highly up-regulated early after germination of S. elegans conidia. This is in agreement with our finding in the EST data mining study, in which a role in toxin production was assigned to cytochrome P450.