Elimination of Airborne Ascospore Inoculum as a Control for Fungal Diseases of Plants

NON-TECHNICAL SUMMARY: The research in Dr. Trail's lab has focused on the fungal pathogen of wheat, Fusarium graminearum. F. graminearum causes head blight of wheat and barley and stalk and ear rot of corn. The severe economic effect of this disease on Midwestern growers is due to the reduced grain yield caused by the fungus, and the presence of the mycotoxin, deoxynivalenol, in contaminated grain. The fungus is spread during the growing season by the forcible ejection of spores (ascospores) from small fungal fruiting bodies (perithecia) on the surface of the crop residues. When the airborne spores land on flowers, they infect and begin to colonize the developing grain. Elimination of the ascospores would result in vast reduction in disease. There are several other fungal diseases of Michigan crops that rely on forcible ascospore discharge to initiate the disease cycle. These include apple scab (Venturia inaequalis), white mold of beans (Sclerotinia sclerotiorum), brown rot of peaches (Monilinia fructicola) and purple spot of asparagus (Stemphylium vesicarium), as well as others. The mechanism of forcible discharge is not clear in any fungus, but we have used Fusarium graminearum as a model system to develop an understanding of how small cellular cannons called asci function to fire their spores. We have made progress in this arena and have identified several methods to inhibit discharge in this fungus. We are beginning to explore the common effects of these methods on other fungi that similarly launch their spores. This project will investigate the mechanism of forcible ascospore discharge and how the phenomenon can be inhibited. MAES delineated 5 target areas that are the major research foci for the experiment station. This research directly impact 3 of these areas. Since F. graminearum produces two major mycotoxins, zearalenone and deoxynivalenol, elimination of the disease would result in better food safety and health to the people of Michigan. In addition, the severe economic consequences of the disease through low grain weight and mycotoxin contamination, which impact grain value, would be reduced by this research, enhancing the profitability of the grain. Finally, knowledge of how to control ascospore discharge could be potentially applied to all pathogens which rely on this dispersal mechanism. This information would help to control the spread of invasive pathogens.

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APPROACH: Objective 1. Understand the mechanism of forcible ascospore discharge in Fusarium graminearum. This ongoing project uses genomics, genetics and physiology to understand the structure of the ascus and the physiology of discharge. Gene knockout experiments are performed routinely in our laboratory to examine the effect of individual candidate genes on ascospore discharge. We are examining calcium, potassium and chloride channels, using gene disruption and localization of proteins. In addition, we are examining the proteins of the cytoskeleton for function in discharge using gene disruption, domain mutagenesis and localization. <P>Objective 2. Apply the model of F. graminearum to the apple scab pathogen, V. inaequalis, to determine how it differs and how it is similar. In collaboration with Dr. Kerik Cox at Cornell University, we are pursuing funding for sequencing of the genome of V. inaequalis. We plan to use homologs of genes discovered in F. graminearum to determine their effect on discharge in the apply scab pathogen, which is a somewhat distant relative and has a different ascus structure. <P>Objective 3. Develop discharge inhibitors that can work on these organisms. We have an ongoing screening process to assess the effect of various compounds on ascospore discharge in F. graminearum. We are developing similar assays in V. inaequalis and several other pathogens. Initially, we will focus on V. inaequalis as a second system to manipulate genetically to determine the similarities between these two scab pathogens. We are seeking funding for an apple scab genome project. We are adapting an Agrobacterium-based transformation system for this pathogen to use RNAi based gene silencing. Once we identify genes and inhibitors that arrest ascospore discharge, we will work with a chemist to optimize these compounds for efficient application to agricultural conditions. <P>Objective 4. Apply the use of these inhibitors to Stemphylium on asparagus and Sclerotinia sclerotiorum on bean. We are developing these methods for testing inhibition of discharge on these organisms. Taking the models of the two scab pathogens we will try to apply similar controls to these destructive pathogens. S. sclerotiorum is difficult to manipulate genetically and Stemphylium does not have genomic resources, so these will be investigated by identifying effective compounds and developing their use for crops affected by these pathogens. <P>

PROGRESS: 2007/01 TO 2007/12 <BR>
Fusarium graminearum produces head blight disease on wheat. The sexual cycle is a crucial component of head blight epidemiology, as forcibly-discharged ascospores serve as the primary inoculum. Using GeneChips, a developmental time course was performed in culture, from vegetative hyphae to mature perithecia with multiseptate ascospores. Time-points represent the development of the major cell-types comprising the mature perithecium. The majority of the 17,830 G. zeae probesets, 78%, were expressed during at least one of the developmental stages; 12% of these are likely to be specific to sexual development. Analysis of the 162 predicted ion transporter genes is reported in detail, due to their association with perithecium function. Expression patterns of the MirA-type siderophores, chloride channels, P-type ATPases and potassium transporters show some specialization in regard to developmental stage. This is the first whole-genome analysis of differential transcript accumulation during sexual development in a filamentous fungus. We also performed a time-course of wheat stem colonization by Fusarium grminearum by inoculation of the head and following fungal progression down the stem. Using this relatively uniform plant tissue, it was possible to separate the hyphae at the front, which are mainly vegetative, from those further back which begin to colonize radially and finally develop perithecium initials at the epidermis. The time-course of gene expression roughly parallels that of sexual development in culture. Cch1, a putative voltage-gated calcium ion channel, was investigated for its role in ascus development in F. graminearum. Gene replacement mutants of CCH1 were generated and found to have asci which did not forcibly discharge spores, although morphologically ascus and ascospore development in the majority of asci appeared normal. Additionally, mycelial growth was significantly slower and sexual development was slightly delayed in the mutant; mutant mycelia showed a distinctive fluffy morphology; and no cirrhi were produced. Wheat infected with ƒ´cch1 mutants developed symptoms comparable to wheat infected with the wild-type, however, the mutants showed a reduced ability to protect the infected stalk from colonization by saprobic fungi. Transcriptional analysis of gene expression in mutants using the Affymetrix Fusarium microarray showed 2449 genes with significant, two-fold or greater changes in transcript abundance across a developmental series. This work extends the role of CCH1 to forcible spore discharge in F. graminearum, and suggests that this channel has subtle effects on growth and development.
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IMPACT: 2007/01 TO 2007/12<BR>
This is the first time the genes unique to spore production have been identified in any filamentous fungi. Filamentous fungi constitute the vast majority of plant disease organisms. Further understanding of the function of these genes is essential to developing novel controls for plant disease. The work also comprises the first identification of genes involved in regulation of ascospore discharge. Numerous fungal pathogens important to US agriculture require forcible ascospore discharge to disperse the primary disease inoculum. Understanding how spores are launched will lead to the identification of chemicals that may be used to controling the dispersal of inoculum.
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PROGRESS: 2006/01/01 TO 2006/12/31<BR>
Lipid accumulation and storage is vital to survival of all organisms. Stored lipids are then used for development. In fungi, lipids are stored in vegetative hyphae and spores as lipid bodies. The wheat pathogen, Gibberella zeae, stores lipids mainly as triacylglycerides (TAG) in anticipation of sexual development. We have characterized the process of lipid accumulation and utilization in association with perithecium development in culture and leading up to perithecium development in planta. We characterized and quantified lipids and water soluble sugars during the initiation and development of perithecia. We next examined gene expression patterns for genes associated with lipid biosynthesis and degradation using data collected from Affymetrix GeneChips. Information gathered from these studies indicates an essential role for lipids in the formation of perithecia. Prior to this study it had been observed that large amounts of fats accumulated immediately following perithecium induction in culture. 1H-NMR analysis of neutral lipids from induced cultures collected at the early stages of perithecium development indicated that TAG were the major stored metabolite in dikaryotic hyphae (those that will produce the perithecia). We examined the transcript accumulation for a discrete set of metabolic genes representing pathways important to lipid accumulation and degradation. Genes representing enzymes related to glycolysis, pentose phosphate pathway, tricarboxylic acid cycle, fatty acid biosynthesis, TAG synthesis and £]-oxidation were identified. The normalized transcript data output was used to establish apparent patterns of expression for TAG storage and degradation through developmental time courses in planta and in culture. In general, accumulation of transcripts for genes associated with TAG generation at the initiation of sexual development was opposite the accumulation for genes associated with TAG breakdown, which occurred during perithecium development.
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IMPACT: 2006/01/01 TO 2006/12/31<BR>
The results of this study clearly show that the head blight fungus must accumulate significant lipid resources from the host in order to produce the following year's inoculum. The accumulation of lipid is most likely due to the transfer of sucrose from the host to the fungus during pathogenesis and is substantially complete by the time the plant is senesced. This is contrary to widespread belief that the fungus invades as a saprophyte and then can produce perithecia, an approach which probably plays little role in the epidemiology of the disease.
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PROGRESS: 2005/01/01 TO 2005/12/31<BR>
Worldwide, one of the most devastating pathogens of small grains is the head blight fungus, Gibberella zeae. Ascospore-laden perithecia of this fungus develop on mature cereal crops and crop debris and provide the primary inoculum of the disease. This year we finished a study to elucidate the process of colonization of wheat tissue, which leads to perithecium production. Stems were systemically and extensively colonized following inoculation of the wheat head. Haploid mycelia moved down the vascular system and pith and then colonized the stem tissue radially. Dikaryotic hyphae developed at two distinct stages: in the xylem, in support of radial hyphal growth and in the chloremchyma, in support of perithecium development. Perithecium formation was initiated in association with stomates and silica cells. Vascular occlusions prevented mycelia from colonizing the stem in 25% of inoculated plants. Vascular occlusions are an important component of resistance for many plants that are hosts to vascular pathogens and may also be an important component of resistance to FHB for wheat. In addition, we elucidated the biomechanical aspects of ascospore discharge. Since wind speed drops to zero at a surface, forced ejection should facilitate spore dispersal. But for tiny spores, with low mass relative to surface area, high ejection speed yields only a short range trajectory, so pernicious is their drag. Thus, achieving high speeds requires prodigious accelerations. In G. zeae, we determined the launch speed and kinetic energy of ascospores shot from perithecia, and the source and magnitude of the pressure driving the launch. We asked whether the pressure inside the ascus suffices to account for launch speed and energy. Launch speed was 34.5 m s-1, requiring a pressure of 1.54 MPa and an acceleration of 870,000 g-- the highest acceleration reported in a biological system. This analysis allows us to discount a major component of the epiplasmic fluid, mannitol, as having a key role in driving discharge, and supports the role of potassium ion flux in the mechanism.
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IMPACT: 2005/01/01 TO 2005/12/31<BR>
We have shown that colonization of wheat vegetation prior to grain harvest in the field is an important step in perithecium development. Vegetation may become colonized by means of stem-base infections and head infections, allowing the head blight pathogen to establish itself prior to saprophytic invasion by other organisms. Strategies to control or eliminate inoculum in the field should focus on slowing or reducing the colonization of wheat vegetation and reducing sporulating structures on debris surfaces. An obvious target for the reduction of vegetative colonization is Type II resistance mechanisms. Reducing sporulation on debris will require a further understanding of the factors that initiate sporulation. An understanding of these processes will aid in measures to reduce potential inoculum production and survival by this fungal plant pathogen.
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PROGRESS: 2004/01/01 TO 2004/12/31<BR>
This year we submitted a manuscript which is a significant contribution to our understanding of fungal development in planta, particularly in regard to function of dikaryotic hyphae. Staining with acridine orange revealed that wide (dikaryotic) hyphae develop in two different paths, depending on their location in the plant. They may give rise to perithecia or give rise to monokaryotic hyphae that continue the colonization process. This result supports our previous finding that wide hyphae are a stage of development unique from thin-infecting hyphae and fungal fruiting bodies. We proposed to identify the major fungal lipids that accumulate prior to perithecium development. This objective has been completed in culture for both wild type parent (PH-1) and the developmental mutant 123C-44, which does not accumulate lipids. In planta, the objective has been completed for PH-1 with results pending for 123C-44. The major fatty acids stored in the wide hpyae in culture were C18:2, C18:1 and C:18 saturated, mainly as triacylglycerides. Cultures induced to produce wide hyphae had nearly twice the fatty acids per dry weight mycelium that uninduced cultures had. The same profile was found with hyphae grown in the wheat stems. No fatty acids were produced in planta by the mutant. My laboratory generated a microarray based on the ESTs generated by Trail, and colleagues. We have completed experiments using these arrays to identify genes showing differential patterns of expression during development. The array represents about 18% of the genes in the genome. Compared with vegetative mycelia, 493, 450, and 326 cDNAs were differentially expressed in 4-day, 5-day and 6-day perithecia, respectively. 109 cDNAs were up-regulated in all 3 perithecial samples compared to vegetative mycelia, in which 70% were specific to the perithecia library. Based on their putative identities, cDNAs that were up-regulated in all three perithecia samples represented genes involved in lipid catabolism, amino acid metabolism and transportation, protein transportation, post-translationally modification, and genes encoding cell wall proteins. Up-regulated genes involved in lipid catabolism included those important for fatty acid elongation, fatty acid oxidation, and metabolisms of membrane phospholipids. These studies give us a handle on genes used for nutritional support for perithecium development.
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IMPACT: 2004/01/01 TO 2004/12/31<BR>
We are moving closer to our goal of understanding inoculum formation and dispersal. we have identified candidate genes, which, if inhibited will reduce the primary inoculum in the field. We have identified a resistance mechanism. These should result in important targets for fungus control.
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PROGRESS: 2003/01/01 TO 2003/12/31<BR>
Gibberella zeae causes head blight of wheat and barley and stalk rot and ear rot of corn. As conventional control measures have not produced effective control of this devastating pathogen, a deeper understanding of the life cycle and biolgy of the fungus is necessary. Our long-term goal is to understand the production and spread of inoculum for this fungal disease. Toward that end, this year we have completed the characterization of fungal growth and sexual development in planta. We now know that the fungus infects and spreads using distinct hyphal types through the various plant tissues. We have identified an effective plant resistance response to the fungus that stops spread within the plant. We have characterized this phenomenon in a susceptible variety. We obtained funding for the genomic sequence of the fungus last year and this year we have begun to annotate it and use it to identify genes important to spore dispersal. 10 genes associated with this function have been disrupted in the fungus and one of them is a proven pathogenicity factor. We have established a microarray based on sequenced genes (ESTs) and used it to identify approximately 100 genes uniquely expressed in the sexual stage. We have just obtained funding for a genomic microarray chip and funds to use it to explore the fungus-host interaction and fungal sexual development.
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IMPACT: 2003/01/01 TO 2003/12/31<BR>
We are moving closer to our goal of understanding inoculum formation and dispersal. we have identified candidate genes, which, if inhibited will reduce the primary inoculum in the field. We have identified a resistance mechanism. These should result in important targets for fungus control.
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PROGRESS: 2002/01/01 TO 2002/12/31<BR>
Fungi have evolved many ways to disperse their spores. Spore dispersal is particularly important to the fungu Gibberella zeae which causes head blight of wheat and barley. To initiate the disease, this fungus fires it sexual spores (the ascospores) into the air, where they are carried to the highly susceptible flowers. We are examining the mechanism of forcible discharge of ascospores in this fungus. We have examined the role for the sugar-alcohol, mannitol, in generating the force to fire these spores. This year we have isolated and characterized the enzyme that produces mannitol, mannitol dehydrogenase and cloned the gene. Preliminary experiments to specifically mutate this gene in the fungus resulted in a partially active enzyme. We are attempting therefore, to isolate a larger clone of the gene from a genomic library. We had previously isolated a mutant strain that does not forcibly discharge it's spores, but develops normally. We have isolated a portion of the mutated gene from this strain and sequence comparisons with the genetic sequences in GenBank indicate that it is not highly homologous with any known genes. We are in the process of cloning the entire gene.
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IMPACT: 2002/01/01 TO 2002/12/31<BR>
Gibberella zeae is dependent on the forcible discharge of its ascospores to initiate a new disease cycle each spring. The identification of possible modes of elimination of this new inoculum source would greatly reduce the impact of the head blight disease. This research is geared towards understanding the phenomenon of spore dispersal with the goal of developing new modes of control.
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PROGRESS: 2001/01/01 TO 2001/12/31<BR>
Gibberella zeae (anamorph Fusarium graminearum) causes head scab of wheat and barley and stalk rot and ear rot of corn. As conventional control measures have not produced effective control of this devastating pathogen, a deeper understanding of the life cycle and biology of the fungus is necessary. Our long-term goal is to understand the production and spread of inoculum for head scab, and the role of the two types of spores (sexual and asexual) in completing the disease cycle. Perithecia, containing the sexual spores, form on crop debris left in the field. We have shown that specific cell types of the wheat plant are infected by mycelia, which the give rise to the perithecia. We have isolated mutants that do not produce perithecia. Using strains with mutations involved in initial stages of perithecium production or lacking perithecia, we can begin the genetic and physiological analysis of this early phase on inoculum production. Our approach is twofold: to combine basic, laboratory research with field research aimed at the ultimate control of inoculum production. Our research combines studies of the basic biology of G. zeae with behavior of the fungus in the field. These two approaches will result in novel approaches to interruption of the disease cycle.
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IMPACT: 2001/01/01 TO 2001/12/31<BR>
Studies on the development and mechanisms of dispersal of the inoculum will provide valuable insight into effective control procedures for this disease. An understanding how field colonized vegetative host tissue supports perithecium production over the course of 1 year may lead to a change is screening for plant resistance. Identification of genes and gene products involved early in perithecium initiation and development will provide targets for control.
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PROGRESS: 2000/01/01 TO 2000/12/31<BR>
Gibberella zeae (anamorph, Fusarium graminearum), the head blight pathogen of wheat and barley, infects during crop flowering. Therefore studies focused on the formation and distribution of the inoculum for flower infection will lead to novel and effective means of control. Ascospores, formed and forcibly discharged from perithecia, are the primary inoculum of this disease. We have focused our efforts on understanding the production and distribution of these propagules. We collected wheat and corn stubble from commercial fields year-round from 1997 to 2000 to evaluate the timing of perithecium formation in the field. Results indicate that perithecium formation is limited by average daily temperatures below 9 C and that corn stubble may be the predominant substrate for perithecium formation in G. zeae in Michigan. The data did not show a limiting high temperature for perithecium formation. We have generated 5000 tagged mutants in G. zeae by insertional mutagenesis using a plasmid conferring hygromycin resistance. These mutants were screened for loss of the ability to forcibly discharge ascospores from perithecia. One mutant has been recovered that forms morphologically normal perithecia, but is discharge minus. Genetic analysis indicates this mutation is tagged with the plasmid. We are currently isolating the gene that has been mutated to determine its function.
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IMPACT: 2000/01/01 TO 2000/12/31<BR>
Information on weather conditions that affect generation of the primary inoculum for the head blight disease can be used in development of prediction systems for head blight epidemics. An understanding of the conditions under which inoculum is formed and disseminated is critical to developing effective means of control for this devastating pathogen.
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PROGRESS: 1999/01/01 TO 1999/12/31<BR>
We are studying the production and distribution of the primary inoculum head blight of wheat and barley. Gibberella zeae, the causal agent of this disease, produces perithecia in the field on crop debris. Ascospores are produced in fruiting bodies, called perithecia, on the crop debris left in the field after harvest. The spores are forcibly discharged into the air where they are carried to infect the next year's flowers. Little is known of the process of formation of perithecia on crop debris, the timing of formation, or the mechanism of forcible discharge of ascospores. We had three objectives to study these issues. Our first objective was to characterize the pattern of colonization of stalk tissue in the mature infected plant, as this tissue will become the debris and eventually yield perithecia. Our second objective was to continue the characterization of timing and appearance of mature perithecia on field debris. Our final objective was to screen 5000 random mutants of G. zeae, created in our lab, for loss of the ability to discharge ascospores. An understanding of the production and distribution of inoculum is vital to designing strategies for disease control. We have collected stalks from plants showing symptoms in naturally infected and inoculated fields. Tissue samples have been removed the nodes and internodal regions of these plants, fixed and embedded in paraffin for histological examination. Preliminary data suggests that mycelia that colonize the epidermal cells before harvest may be important overwintering tissue for the fungus and may give rise to the perithecia in the spring. The embedded samples will be sectioned and examined microscopically to formulate a picture of the infection pattern of the tissue that will become the crop debris. The pattern of appearance of the perithecia on wheat and corn over the last 3 years. In the last 2 years flowering has been quite early, before our collections showed perithecium production in the field. In all three years the disease incidence has been low. We will continue to collect monthly through at least 2 more years. Data will be added as the samples are analyzed. We have generated over 5000 insertional mutants of G. zeae and screened over 3500 mutants to date for loss of discharge. Twenty of the isolates have shown a loss of discharge in preliminary trials. These putative mutants need to be tested for stability of the phenotype through meiosis. We are in the process of this analysis and continue to screen other mutants for possible loss of discharge. We have identified mannitol as the sole simple sugar found in the ascus exudates, discharged from the perithecia along with the ascospores. We hypothesize that mannitol is crucial in generation of hydrostatic pressure within the ascus. We are currently attempting to genetically disrupt the gene for mannitol dehydrogenase, the mannitol-generating enzyme.
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IMPACT: 1999/01/01 TO 1999/12/31<BR>
Gibberella zeae is the causal agent of the economically devastating wheat and barley scab disease. My research will result in novel methods for control of the primary inoculum of this disease.