The analysis of gene transcripts associated with conidiation in the insect pathogenic fungus, Metarhizium anisopliae /

Conidia of the insect pathogenic fungus, Metarhizium anisopliae play an important role in pathogenicity because they are the infective propagules that adhere to the surface of the insect, then germinate and give rise to hyphal penetration of the insect cuticle. Conidia are produced in the final stages of insect infection as the mycelia emerge from the insect cadaver. The genes associated with conidiation have not yet been studied in this fiingus. hi this study we used the PCR-based technique, suppression subtractive hybridization (SSH) to selectively amplify conidial-associated genes in M. anisopliae. We then identified the presence of these differentially expressed genes using the National Center for Biotechnology Information database. One of the transcripts encoded an extracellular subtilisin-like protease, Prl, which plays a fundamental role in cuticular protein degradation. Analysis of the patterns of gene expression of the transcripts using RT-PCR indicated that conidial-associated cDNAs are expressed during the development of the mature conidium. RT-PCR analysis was also performed to examine in vivo expression of Prl during infection of waxworm larvae {Galleria mellonelld). Results showed expression of Prl as mycelia emerge and produce conidia on the surface of the cadaver. It is well documented that Prl is produced during the initial stages of transcuticular penetration by M. anisopliae. We suggest that upregulation of Prl is part of the mechanism by which reverse (from inside to the outside of the host) transcuticular penetration of the insect cuticle allows subsequent conidiation on the cadaver.

Cloning, sequencing and characterization of a chitin synthase gene fragment from the fungus Phascolomyces articulosus /

Phascolomyces articulosus genomic DNA was isolated from 48 h old hyphae and was used for amplification of a chitin synthase fragment by the polymerase chain reaction method. The primers used in the amplification corresponded to two widely conserved amino acid regions found in chitin synthases of many fimgi. Amphfication resulted in four bands (820, 900, 1000 and 1500 bp, approximately) as visualized in a 1.2% agarose gel. The lowest band (820 bp) was selected as a candidate for chitin synthase because most amplified regions from other fimgi so far exhibited similar sizes (600-750 bp). The selected fragment was extracted from the gel and cloned in the Hinc n site of pUC19. The derived plasmid and insert were designated ^\5C\9'PaCHS and PaCHS respectively. The plasmid pUC19-PaC/fS was digested by several restriction enzymes and was found to contain BamHl and HincU sites. Sequencing of PaCHS revealed two intron sequences and a total open reading frame of 200 amino acids. The derived polypeptide was compared with other related sequences from the EMBL database (Heidelberg, Germany) and was matched to 36 other fiilly or partially sequenced fimgal chitin synthase genes. The closest resemblance was with two genes (74.5% and 73.1% identity) from Rhizopus oligosporus. Southern hybridization with the cloned fragment as a probe to the PCR reaction showed a strong signal at the fragment selected for cloning and weaker signals at the other two fragments. Southern hybridization with partially digested Phascolomyces articulosus genomic DNA showed a single band. The amino acid sequence was compared with sequences from other chitin synthase gene classes using the CLUSTALW program. The chitin synthase fragment from Phascolomyces articulosus was initially grouped in class n along with chitin synthase fragments from Rhizopus oligosporus and Phycomyces blakesleeanus which also belong to the same class, Zygomycetes. Bootstrap analysis using the neighbor-joining method available by CLUSTALW verified such classification. Comparison of PaCHS revealed conservation of intron positions that are characteristic of chitin synthase gene fragments of zygomycetous fungi.

Incidence of resistance to benzimidazole fungicides in a field population of Venturia inaequalis /

The allele-specific polymerase chain reaction (PCR) was used to screen for the presence of benomyl resistance, and to characterize their levels and frequencies in field populations of Venturia inaequalis during two seasons. Three hundred isolates of V. inaequalis were collected each season from infected leaves of MalusX domestica. Borkh c.v. Mcintosh. The trees used were sprayed in the year prior to collection with five applications of benomyl, its homologue Azindoyle, or water. Monoconidial isolates of V. inaequalis were grown on 2% potato dextrose agar (PDA) for four weeks. Each isolate was taken from a single lesion from a single leaf. Total genomic DNA was extracted from the four week old colonies of V. inaequalis, prepared and used as a template in PCR reactions. PCR reactions were achieved by utilizing allele-specific primers. Each primer was designed to amplify fragments from a specific allele. Primer Vin was specific for mutations conferring the ben^^"^ phenotype. It was expected to amplify a 171 bp. DNA fragment from the ben^"^ alleles only. Primers BenHR and BenMR were specific for mutations conferring the ben"" and ben'^'' phenotypes, respectively. They were expected to amplify 172 bp. and 165 bp. DNA fragments from the ben"" and ben"^" alleles, respectively. Of the 953 isolates tested, 414 (69.9%) were benomyl sensitive (ben^) and 179 (30.1%) were benomyl resistant. All the benomyl resistant alleles were ben^"", since neither the ben"" nor the ben"" alleles were detected. Frequencies of benomyl resistance were 23%, 24%, and 23% for the 1997 collections, and were 46%, 26% and 38% for the 1998 collections for benomyl, Azindoyle and water treatments, respectively. Growth assay was performed to evaluate the applicability of using PCR in monitoring benomyl resistance in fungal field populations. Tests were performed on 14 isolates representing the two phenotypes (ben^ and ben^"'' alleles) characterized by PCR. Results of those tests were in agreement with PCR results. Enzyme digestion was also used to evaluate the accuracy and reliability of PCR products. The mutation associated with the ben^"'' phenotype creates a unique site for the endonuclease enzyme Bsh^236^ allowing the use of enzyme digestion. Isolates characterized by PCR as ben^'^'^ alleles had this restriction site for the SsA7l2361 enzyme. The most time consuming aspect of this study was growing fungal isolates on culture media for DNA extraction. In addition, the risk of contamination or losing the fungus during growth processes was relatively high. A technique for extracting DNA directly from lesions on leaves has been used (Luck and Gillings 1 995). In order to apply this technique in experiments designed to monitor fungicide resistance, a lesion has to be homogeneous for fungicide sensitivity. For this purpose, PCR protocol was used to determine lesion homogeneity. One hundred monoconidial isolates of V. inaequalis from 10 lesions (10-conidia/ lesion) were tested for their phenotypes with respect to benomyl sensitivity. Conidia of six lesions were homogeneous, while conidia of the remaining lesions were mixtures of ben^ and ben^ phenotypes. Neither the ben" nor the ben' phenotype was detected.

Aspergillus flavus infections in Galleria mellonella : a pathogen-host model system for the study of emerging diseases

To study emerging diseases, I employed a model pathogen-host system involving infections of insect larvae with the opportunistic fungus Aspergillus flavus, providing insight into three mechanisms ofpathogen evolution namely de novo mutation, genome decay, and virulence factoracquisition In Chapter 2 as a foundational experiment, A. flavus was serially propagated through insects to study the evolution of an opportunistic pathogen during repeated exposure to a single host. While A. flavus displayed de novo phenotypic alterations, namely decreased saprobic capacity, analysis of genotypic variation in Chapter 3 signified a host-imposed bottleneck on the pathogen population, emphasizing the host's role in shaping pathogen population structure. Described in Chapter 4, the serial passage scheme enabled the isolation of an A. flavus cysteine/methionine auxotroph with characteristics reminiscent of an obligate insect pathogen, suggesting that lost biosynthetic capacity may restrict host range based on nutrient availability and provide selection pressure for further evolution. As outlined in Chapter 6, cysteine/methionine auxotrophy had the pleiotrophic effect of increasing virulence factor production, affording the slow-growing auxotroph with a modified pathogenic strategy such that virulence was not reduced. Moreover in Chapter 7, transformation with a virulence factor from a facultative insect pathogen failed to increase virulence, demonstrating the necessity of an appropriate genetic background for virulence factor acquisition to instigate pathogen evolution.

Primary characterization of genes involved in the colony development of the insect pathogenic fungus Metarhizium anisopliae /

Strain improvement of the insect pathogenic fungus Metarhizium anisopUae is necessary to increase its virulence towards agricultural pests and thus improve its commercial efficacy. Nevertheless, the release of genetically modified conidia in crop fields may negatively affect the ecosystem. Controlling conidiation is a potential means of limiting the release of engineered strains since conidia are the infective propagules and the means of dispersal. The purpose of this study was to research the colony development of M. anisopUae to identify potential targets for genetic manipulation to control conidiation. Following Agrobacterium tumefaciem insertional mutagenesis, phenotypic mutants were characterized using Y-shaped adaptor dependent extension PCR. Four of 1 8 colony development recombinants had T-DNA flanking sequences with high homology to genes encoding known signaling pathway proteins that regulate pathogenesis and/or asexual development in filamentous fungi. Conidial density counts and insect bioassays suggested that a Serine/Threonine protein kinase COTl homolog is not essential for conidiation or virulence. Furthermore, a choline kinase homolog is important for conidiation, but not virulence. Finally, the regulator of G protein signaling CAG8 and a NADPH oxidase NoxA homolog are necessary for conidiation and virulence. These genes are candidates for further investigation into the regulatory pathways controlling conidiation to yield insight into promising gene targets for biocontrol strain improvement.

Gypsy moths (Lymantria dispar) in the Niagara Region : population density variation, introduction of an entomopathogenic fungus (Entomophaga maimaiga) and occurrence of nuclear polyhedrosis virus

The gypsy moth, Lymantria dispar, a major defoliator of broad leaf trees, was accidentally introduced into North America in 1869. Much interest has been generated regarding the potential of using natural pathogens for biological control of this insect. One of these pathogens, a highly specific fungus, Entomophaga maimaiga, was accredited with causing major epizootics in populations of gypsy moth across the north-eastern United States in 1989 and 1990 and is thought to be spreading northwards into Canada. This study examined gypsy moth population densities in the Niagara Region. The fungus, .E.. maimaiga, was artificially introduced into one site and the resulting mortality in host populations was noted over two years. The relationship between fungal mortality, host population density and occurrence of another pathogen, the nuclear polyhedrosis virus (NPV), was assessed. Gypsy moth population density was assessed by counting egg masses in 0.01 hectare (ha) study plots in six areas, namely Louth, Queenston, Niagara-on-the-Lake, Shorthills Provincial Park, Chippawa Creek and Willoughby Marsh. High variability in density was seen among sites. Willoughby Marsh and Chippawa Creek, the sites with the greatest variability, were selected for more intensive study. The pathogenicity of E. maimaiga was established in laboratory trials. Fungal-infected gypsy moth larvae were then released into experimental plots of varying host density in Willoughby Marsh in 1992. These larvae served as the inoculum to infect field larvae. Other larvae were injected with culture medium only and released into control plots also of varying host density. Later, field larvae were collected and assessed for the presence of .E.. maimaiga and NPV. A greater proportion of larvae were infected from experimental plots than from control plots indicating that the experimental augmentation had been successful. There was no relationship between host density and the proportion of infected larvae in either experimental or control plots. In 1992, 86% of larvae were positive for NPV. Presence and intensity of NPV infection was independent of fungal presence, plot type or interaction of these two factors. Sampling was carried out in the summer of 1993, the year after the introduction, to evaluate the persistence of the pathogen in the environment. Almost 50% of all larvae were infected with the fungus. There was no difference between control and experimental plots. Data collected from Willoughby Marsh indicated that there was no correlation between the proportion of larvae infected with the fungus and host population density in either experimental or control plots. About 10% of larvae collected from a nearby site, Chippawa Creek, were also positive for .E.. maimaiga suggesting that low levels of .E.. maimaiga probably occurred naturally in the area. In 1993, 9.6% of larvae were positive for NPV. Again, presence or absence of NPV infection was independent of fungal presence plot type or interaction of these two factors. In conclusion, gypsy moth population densities were highly variable between and within sites in the Niagara Region. The introduction of the pathogenic fungus, .E.. maimaiga, into Willoughby Marsh in 1992 was successful and the fungus was again evident in 1993. There was no evidence for existence of a relationship between fungal mortality and gypsy moth density or occurrence of NPV. The results from this study are discussed with respect to the use of .E.. maimaiga in gypsy moth management programs.

Protein subcellular targeting inTrichoderma aggressivum f. aggressivum

Trichoderma aggressivum f. aggressivum is a filamentous soil fungus. Green mold disease of commercial mushrooms caused by this species in North America has resulted in millions of dollars in lost revenue within the mushroom growing industry. Research on the molecular level of T aggressivum have jus t begun with the goal of understanding the functions of each gene and protein, and their expression control. Protein targeting has not been well studied in this species yet. Therefore, the intent of this study was to test the protein localization and production levels in T aggressivum with green fluorescent protein (GFP) with an intron and tagged with either nuclear localization signal (NLS) or an endoplasmic reticulum retention signal (KDEL). Two GFP constructs (with and without the intron) were used as controls in this study. All four constructs were successfully transferred into T aggressivum and all modified strains showed similar growth characteristics as the wild type non-transformed isolate. GFP expression was detected from all modified T aggressivum with confocal microscopy and the expression was similar in all four strains. The intron tested in this study had no or very minor effects as GFP expression was similar with or without it. The GFP signal increased over a 5 day period for all transformants, while the GFP to total protein ratio decreased over the same period for all transformants. The GFP-KDEL transformant showed similar protein expression level and localization as did the control transformant lacking the KDEL retention signal. The GFP-NLS transformant similarly failed to localize GFP into nucleus as fluorescence with this strain was virtually identical to the GFP transformant lacking the NLS. Thus, future research is required to find effective localization signals for T aggressivum.

Starvation Induces Expression of the Plant-Adhesin Gene, Mad2, of the Entomopathogenic Fungus Metarhizium robertsii.

Metarhizium robertsii is an entomopathogenic fungus that is additionally plant rhizosphere competent. Two adhesin-encoding gens, Mad1 and Mad2, are involved in insect pathogenesis or plant root colonization, respectively. This study examined differential expression of the Mad genes for M robertsii grown on a variety of insectand plant-related substrates. Mad1 was up regulated in response to insect cuticles and up regulation of Mad2 resulted from root exudates, tomato stems and non-preferred carbohydrates. A time course analysis that compared water, minimal media, and nutrient rich broth revealed Mad2 gene expression increased as nutrient availability decreased. The regulation of Mad2 compared to known stress-related genes (Hsp30, Hsp70 and ssgA) under various stresses (nutrient, pH, osmotic, oxidative, temperature) revealed Mad2 to be generally up regulated by nutrient starvation only. Examination of the Mad2 promoter region revealed two copies of a stress-response element (S TRE) known to be regulated under the general stress response pathway.

Letters Patent Incorporating The Ontario Grape Growing and Wine Manufacturing Company

The patent incorporates the Ontario Grape Growing and Wine Manufacturing Company for the purpose "for the manufacture of wine within the province of Ontario" and is recorded as no. 85 on June 9, 1873.