1 The Pennsylvania State University The Graduate School Department of Plant Pathology FUSARIUM HEAD BLIGHT DISEASE DEVELOPME T A D MYCOTOXI ACCUMULATIO I WHEAT A Dissertation in Plant Pathology by Katelyn Tilley Willyerd 2009 Katelyn Tilley Willyerd Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy August 2009
2 The dissertation of Katelyn Tilley Willyerd was reviewed and approved* by the following: Gretchen A. Kuldau Associate Professor of Plant Pathology Dissertation Adviser Chair of Committee Douglas D. Archibald Research Associate in Agricultural Analytical Chemistry Erick D. De Wolf Associate Professor of Plant Pathology Kansas State University Special Member Maria del Mar Jimenez-Gasco Assistant Professor of Plant Pathology Gary W. Moorman Professor of Plant Pathology Henry K. Ngugi Assistant Professor of Plant Pathology Frederick E. Gildow Professor and Head of Department of Plant Pathology *Signatures are on file in the Graduate School
3 iii ABSTRACT Fusarium graminearum, causal agent of Fusarium Head Blight (FHB) of wheat, causes yield losses and contaminates grain with mycotoxins, most commonly deoxynivalenol (DON). FHB is one of the most destructive and economically important plant diseases and is found in all wheat-growing regions of the world. DON is the most widely encountered mycotoxin by humans in the world. This toxin binds to eukaryotic ribosomes and inhibits protein synthesis in wheat, humans and animals. During acute exposure DON can cause cell death, vomiting and suppress the immune system. Annual FHB epidemics and DON levels are extremely influenced by the environment. The effects of environment on the relationships between fungal growth, disease development and DON accumulation are not fully understood. Without this basic knowledge, FHB and DON prevention, control and mitigation will continue to be a problem for all involved in the wheat industry and consumers. The purpose of this dissertation was to characterize FHB disease development and mycotoxin accumulation during the grain development stages in wheat. A two-year field experiment was designed to study the effects of infection-timing and cultivar on disease severity, kernel damage and accumulation of DON. Three winter wheat cultivars, with different degrees of susceptibility to FHB, were planted in a splitplot design. Misting treatments were designed to facilitate infections during anthesis and/or late-milk stages. All plots were inoculated with F. graminearum macroconidia prior to each misting treatment to ensure the presence of the pathogen. Disease severity of each subplot was assessed during the dough stages, while Fusarium-damaged kernels (FDK) and DON accumulation were measured post-harvest. Misting treatment, cultivar
4 iv and their interactions were significant factors for severity, FDK and DON. This study confirmed that infections during both anthesis and grain-fill contributed to symptoms and DON levels. Infections during grain-fill alone contributed to DON accumulation (> 2ppm) and had little effect on symptoms. Response to infection-timing was found to be cultivar-specific. Infection-timing and host genotype interactions play significant roles in disease development and mycotoxin contamination of wheat. Future work should incorporate a wider range of wheat cultivars so as to gain more information on the effect of infection-timing. The second objective of this work was to develop a method to simultaneously detect DON and fungal biomass in wheat heads, as the relationship between toxin production and fungal growth is not fully understood. By analyzing single wheat florets from infected wheat heads, infection and toxin accumulation patterns could be studied. Deoxynivalenol was extracted from FHB-affected single florets with acetonitrile-water. Ergosterol, a fungal-specific sterol found in the cell membrane, was used as a biomarker for fungal biomass. Ergosterol was removed from the same wheat florets through saponification and extraction with hexane. Toxin and ergosterol extracts were combined and analyzed via gas chromatography with electron capture detection (GC-ECD). This method was also designed to detect deoxynivalenol-3-glucoside (DON-gluc), a conjugated mycotoxin, which forms in planta. Retention times were confirmed with analytical standards and standard curves were created to estimate concentrations of these in floret samples. The limits of quantification were 0.005, and 0.100ng/µl for DON, ergosterol and DON-gluc, respectively. The extraction protocols and GC-ECD method are tools with the potential to study trichothecene accumulation and fungal colonization of many important agricultural commodities.
5 v The final goal was to use the GC-ECD method to study the effects of temperature and cultivar on fungal biomass and DON accumulation in wheat heads. Two spring wheat cultivars were used in this study: Alsen (moderately resistant) and Wheaton (susceptible). A central spikelet was inoculated during mid-anthesis. Plants were incubated at 15 or 22 o C. Spikelets, each containing two florets, were harvested at 2, 4, 6, 8 and 10 days post-inoculation (dpi). One floret was placed on Nash agar to determine F. graminearum incidence. The remaining floret was reserved for GC-ECD analysis. Colonization beyond the inoculated spikelet and DON translocation to florets not colonized by Fusarium were observed by 2dpi. During the early stages of infection, wheat heads in the 22 o C treatment experienced greater Fusarium incidence than those incubated at 15 o C. The interactive effect of cultivar and temperature was significant for both DON and ergosterol accumulation in wheat florets. The resistant cultivar Alsen experienced the highest levels of DON accumulation when incubated at 15 o C, but the least amount of ergosterol. This suggests that stressful conditions, such as resistant host and cool temperatures, may limit Fusarium growth and stimulate DON production during early stages of grain development. Both cultivars produced DON-gluc by 6dpi. Wheaton produced greater levels of DON-gluc than Alsen, especially by 10dpi. This may, in part, explain the lack of DON increase between 8 and 10dpi. Future work may include extending harvest dates to characterize DON, DON-gluc and ergosterol production and accumulation throughout grain development until harvest. The findings of this dissertation contribute to the greater understanding of FHB epidemiology and mycotoxin accumulation in wheat.
6 vi TABLE OF CO TE TS LIST OF FIGURES... viii LIST OF TABLES... xi ACKNOWLEDGEMENTS... xiii Chapter 1. I TRODUCTIO A D THESIS OBJECTIVES... 1 Crop Biology and Significance... 1 Fusarium Head Blight History and Significance... 3 Biology of Fusarium graminearum... 6 Trichothecene Mycotoxins... 9 Disease Symptoms and Signs Fusarium Head Blight Disease Cycle Disease and Mycotoxin Management I. Cultural Practices II. Host Resistance Characteristics III. Fungicides IV. Role of Fertilizers V. Biological Control for FHB Management Environmental Effects on Disease and Mycotoxin Development I. Temperature II. Moisture and Humidity Infection Characteristics within Wheat Heads Summary Thesis Hypotheses Objectives for Research Chapter 2. EFFECTS OF I FECTIO -TIMI G DURI G WHEAT DEVELOPME T O FUSARIUM HEAD BLIGHT A D DEOXY IVALE OL ACCUMULATIO Introduction Materials and Methods Field Design and Treatment Description Inoculations of Field Plots Misting Treatments Data Collection and Statistical Analysis Results Effects of Year Effects of Fixed Factors Pair-wise Comparisons of Cultivar-Treatment Interactions I. Disease Severity II. Fusarium-Damaged Kernels III. Deoxynivalenol Discussion... 50
7 Chapter 3. A EW METHOD TO DETECT DEOXY IVALE OL, DEOXY IVALE OL-3-GLUCOSIDE A D ERGOSTEROL I SI GLE WHEAT FLORETS Introduction Materials and Methods Trichothecene Extraction Ergosterol Extraction Peak Identification and Standard Curves Method Recovery and Hyphal Assessment Sample Derivatization Instrumentation and Analytical Program Settings Results Discussion Chapter 4. DEOXY IVALE OL ACCUMULATIO A D FUSARIUM GRAMI EARUM I FECTIO PATTER S I WHEAT HEADS Introduction Materials and Methods Inoculum Preparation Wheat Production in the Greenhouse Point Inoculations Floret Harvests Sample Preparation and Analysis Statistical Analysis Results Incidence of Fusarium in Wheat Florets Deoxynivalenol Translocation Fungal Biomass Estimated by Ergosterol Deoxynivalenol-3-glucoside Synthesis in Wheat Heads Discussion vii Chapter 5. SUMMARY A D FUTURE DIRECTIO S LITERATURE CITED APPE DIX: ZADOKS SMALL GRAIN GROWTH STAGE SYSTEM
8 LIST OF FIGURES Figure 1-1. A map illustrating wheat acreage planted in the United States during the 2007 growing season (Source: USDA-NASS; accessed 2 April 2009) Figure 1-2. The chemical structure of deoxynivalenol (DON, vomitoxin), a trichothecene mycotoxin produced by Fusarium species, shows the toxic, unstable epoxide moiety Figure 1-3. The schematic demonstrates the disease cycle of Fusarium Head Blight, from inoculum dispersal in the spring to wheat harvest in the late summer Figure 2-1. The schematic above details the field design for this infectiontiming experiment. One replication consisted of four treatment plots (wet-dry, dry-wet, wet-wet, ambient) each with three cultivar subplots (Hop = Hopewell, Tru = Truman, Val = Valor ) Figure 2-2. The photograph depicts the spray inoculation of Fusarium graminearum (10 4 macroconidia/ml) on wheat. All plots were inoculated during anthesis and late-milk stages of wheat head development Figure 2-3. The photograph shows the wooden-framed mist chambers which were placed over the cultivar sub-plots to provide misting at anthesis and/or late milk stages. The plastic-covered moveable greenhouse in the background is signaled by a moisture sensor to cover these experimental plots during rain events. The track, on which the greenhouse moves back and forth, is observable in the right-hand side of this photograph Figure 2-4. This photographic scale, developed by Engle et al. (1998), was used to estimate the percent of Fusarium-damaged wheat kernels per harvested subplot Figure 3-1. Wheat is able to detoxify deoxynivalenol by forming deoxynivalenol-3-glucoside using glycosyltransferase enzymes in planta Figure 3-2. The chemical structure of ergosterol, C 28 H 44 O, is typical of sterols. The molecule is non-polar with the exception of the hydroxyl group at the 3-position Figure 3-3. This method flow chart describes the steps taken to extract from wheat floret tissue, clean and derivatize deoxynivalenol, deoxynivalenol-3-glucoside and ergosterol viii
9 Figure 3-4. A) The GC-ECD chromatogram shows peaks corresponding to deoxynivalenol (DON, min), the internal standard mirex (36.5 min), deoxynivalenol-3-glucoside (DON-Gluc, min) and 4) ergosterol (ERG, min), and B) acylated derivatives of deoxynivalenol, 3-acetyldeoxynivalenol (3ADON, 27.1min) and 15- acetyldeoxynivalenol (15ADON, 26.8min). Chromatogram B has been enlarged and cropped to show peak detail Figure 3-5. The standard curves for deoxynivalenol, deoxynivalenol-3- glucoside and ergosterol depict the linear relationships between compound concentration and peak area Figure 3-6. The GC-ECD chromatogram depicts the mean deoxynivalenol (DON, 21.2 minutes, 0.095ppm) and ergosterol (ERG, 52.7 minutes, ppm) content of F. graminearum mycelium grown in potato dextrose broth. Mirex (37.9, 1.0 ng/µl) was used as an internal standard Figure 4-1. Wheat floret anatomy is depicted in this photograph. Between the lemma and palea, the feathery stigma and yellow anthers of the flower can be observed. (Source: whspik.jpg) Figure 4-2. These photographs depict the single spikelet inoculation protocol. A) A central spikelet is selected and each floret is denoted with a marker. B) The lemma and palea are separated and central spikelet is inoculated with F. graminearum macroconidia during midanthesis. C) The entire wheat head is enclosed in a plastic bag to incubate for 48 hours Figure 4-3. A sample wheat head shows the position of the inoculated central spikelet (denoted 0 ) in relation to the harvested spikelets both above (+1 to +4) and below (-1 to -4) the point of inoculation. While shown in profile here, it should be noted that each wheat spikelet is composed of two florets. In this study one floret was plated on Nash agar and the other was used for chromatographic analysis Figure 4-4. Mean deoxynivalenol concentrations in single florets following point inoculation of floret 0 of moderately-resistant Alsen (A) and susceptible Wheaton (B). Florets were incubated at 15 or 22 o C, harvested 2, 4, 6, 8 and 10 days post-inoculation and estimated by gas chromatography-electron capture detection ix
10 Figure 4-5. Mean ergosterol concentrations in single florets following point inoculation of floret 0 of moderately-resistant Alsen (A) and susceptible Wheaton (B). Florets were incubated at 15 or 22 o C, harvested 2, 4, 6, 8 and 10 days post-inoculation and estimated by gas chromatography-electron capture detection Figure 4-6. Deoxynivalenol-3-glucoside concentration in single florets following point inoculation of floret 0 of moderately-resistant Alsen (A) and susceptible Wheaton (B). Florets were incubated at 15 or 22 o C, harvested 2, 4, 6, 8 and 10 days post-inoculation and estimated by gas chromatography-electron capture detection x
11 xi LIST OF TABLES Table 2-1. Analysis of variance for the fixed effects of infection-timing treatment, wheat cultivar and their interaction on Fusarium Head Blight disease incidence, severity, Fusarium-damaged kernels and deoxynivalenol accumulation in 2006 and Table 2-2. Infection-timing treatment and cultivar have interactive effects on mean Fusarium Head Blight disease severity (%) during the 2006 and 2007 field seasons Table 2-3. Infection-timing treatment and cultivar have interactive effects on mean Fusarium-damaged kernel ratings (%) during the 2006 and 2007 field seasons Table 2-4. Infection-timing treatment and cultivar have interactive effects on mean deoxynivalenol accumulation (ppm) in harvested wheat during the 2006 and 2007 field seasons Table 3-1. Wheat florets were spiked with known concentrations of deoxynivalenol, deoxynivalenol-3-glucoside and ergosterol and recovery (%) following extraction, clean-up and derivatization was calculated Table 4-1. The effects of fixed factors on Fusarium incidence in wheat heads following a point inoculation of resistant and susceptible wheat cultivars, incubation at 22 or 15 o C and harvest at 2,4,6,8,and 10 days post-inoculation Table 4-2: Fusarium incidence for each harvested day post-inoculation, calculated across all cultivars, temperature treatments and individual florets Table 4-3: The effects of fixed factors on DON accumulation in Alsen and Wheaton wheat florets incubated at 15 or 22 o C and harvested by 2, 4,6, 8 or 10 days post-inoculation Table 4-4: Deoxynivalenol accumulation calculated for each cultivar*temperature interaction, across all harvest days and florets Table 4-5: Deoxynivalenol accumulation for each harvested day postinoculation, calculated across all cultivars, temperature treatments and individual florets Table 4-6: The effects of fixed factors on fungal growth and colonization of Alsen and Wheaton wheat florets incubated at 15 or 22 o C and harvested by 2, 4,6, 8 or 10 days post-inoculation, as estimated by ergosterol
12 Table 4-7: Ergosterol accumulation for each harvest day, calculated across all cultivars, temperature treatments and individual florets Table 4-8: Ergosterol accumulation estimated for each cultivar*temperature interaction, across all harvest days and florets Table 4-9: Mean deoxynivalenol-3-glucoside and deoxynivalenol accumulation and the ratio between the two compounds in wheat heads xii
13 xiii ACK OWLEDGEME TS I would like to thank the faculty, staff and students of the Plant Pathology Department for their support and assistance throughout my time as a graduate student. This dissertation is certainly shared by all who contributed their time and expertise to my personal and professional development. I must especially thank Dr. Gretchen Kuldau, my advisor, who has provided me with amazing opportunities here at Penn State and beyond. I have been given a glimpse of how far a passion for science can take me. I also thank Erick De Wolf for his continued guidance throughout my dissertation. Additionally, I d like to acknowledge my committee members for their support of my research: Dr. Douglas Archibald, Dr. David Geiser, Dr. Maria del Mar Jimenez- Gasco, Dr. Gary Moorman and Dr. Henry Ngugi. I must also give credit to Mr. Tim Grove and Dr. Mizuho Nita for their significant contributions to the field study described in Chapter 2. Lastly, I would like to thank the members of the Kuldau lab, past and present who have assisted me with my projects over the past four years. I appreciate contributions of Mr. Adam Blatt to the single floret study in Chapter 4 and the daily moral support I received from Mrs. Nancy Wenner. Personally, I would like to thank my family: John and Nancy Tilley and Kermit and Louise Taggart. They have taught me the value and power of education and the importance of becoming a life-long learner. I credit my family with my introduction to agriculture and plant pathology while growing up on our apple orchard. Finally, I must thank Scott Willyerd, my husband. Without his love, support and hard work in the field, this dissertation would not have been possible.
14 Chapter 1 I TRODUCTIO A D THESIS OBJECTIVES Crop Biology and Significance Wheat (Triticum aestivum) is a member of the Poaceae family and is an important agronomic crop in the United States and around the world. Originating from the Middle East, this grain is a staple food for many people worldwide, and roughly half of the wheat grown in the U.S. is exported (5). The largest importer of U.S. wheat is Japan, according to the United States Department of Agriculture (USDA). According to the National Agricultural Statistic Service, the U.S. produced approximately 2.5 billion bushels of wheat in 2008, a value exceeding 16.5 billion U.S. dollars (174). Kansas leads the country in wheat production (8.9 million acres), generating over $2.5 billion in revenue in 2008 (174). In Pennsylvania, a relatively minor wheat producing state, 185,000 acres of wheat were harvested in the 2008 season. The value of this crop was estimated to be $71.6 million. Most regions of the continental United States produce wheat, but the Great Plains region produces the majority (5) (Figure 1-1). Climate dictates the type of wheat that is grown in specific regions. Winter wheat is sown in the fall and must undergo vernalization during the winter months before it will head, flower and set seed. Winter wheat, accounting for two-thirds of U.S. wheat production, resumes growth in the spring and is harvested in July or August (5). Spring wheat is sown in the spring and is also harvested during mid-summer. Spring wheat is more commonly grown in the Northern Plains, while winter wheat is more prevalent in the Central and Southern Plains. Wheat is also classified according to grain qualities such as protein content, which influences the
15 2 use of the grain. Ali et al. (2000) developed a glossary of wheat types. Hard wheat has high protein content and is used for bread-making and all-purpose flour. Hard red winter wheat, produced in the Great Plains, accounts for 40% of U.S. production and the majority of exports. Hard red spring is also used for bread-making, but is primarily grown in the Northern Plains and the Red River Valley. Durum wheat, grown in North Dakota, has the highest protein content and is ideal for pastas. Soft wheat has lower protein content and is used in making cakes, pastries, cookies, crackers and snack foods. Soft white wheat is mainly grown in the Pacific Northwest and used in bakery goods other than breads. Twenty-five percent of wheat acreage in the Pacific Northwest is irrigated, but it is less profitable to do so in other wheat-growing regions (5). The highyielding soft red winter wheat is grown in areas east of the Mississippi River, including Pennsylvania. In general, wheat is adaptable to environmental conditions; however climate change certainly has the potential to affect the wheat industry. Using climatic models, it was estimated that winter wheat production could drop as much as 18% if the global mean temperature were to increased by 2.5 o C (26).
16 3 Figure 1-1. A map illustrating wheat acreage planted in the United States during the 2007 growing season (Source: USDA-NASS; accessed 2 April 2009). Fusarium Head Blight History and Significance Fusarium Head Blight (FHB) is a costly and destructive disease of wheat and barley and has been a problem sporadically for United States farmers for most of the twentieth century. The disease was first described in 1884 by English scientist W. G. Smith, who identified Fusarium culmorum as the causal agent of a fungal disease of wheat he called scab. In the U.S., FHB was reported during the early 1890 s in the eastern wheat growing regions of the country (3). The disease has been reported in all cereal-growing regions in the world (106). FHB has been associated with 17 different fungal species, but F. culmorum and F. graminearum appear to play a predominate role in wheat infections
17 4 in Europe and North America (120). F. culmorum is favored by the cooler environments of Western Europe and Canada, whereas F. graminearum is favored by the warmer, temperate zones of the United States. A severe epidemic occurred in the upper Midwest and Canada in 1993 and the estimated loss approached one billion dollars (106). From 1992 to 1993 wheat yield dropped by 50 and 40% in North Dakota and Minnesota respectively (183). Scabby kernels constituted up to 70% of the grain harvested from the Northern Plains (106). In addition to low test weight, infected wheat kernels may also have reduced germination rates and seedling vigor (16). Epidemics are associated with weather conditions, specifically the timing of rainfall, heavy dews and humidity, during anthesis and early grain-fill stages. The epidemic of 1993 was no exception, as rainfall during that July was higher than the normal average (106). Conservation tilling practices were also quite common in this area leading to an increased supply of available inoculum at the soil surface. Nearly one fourth of wheat growers report practicing conservation tillage (5). The availability of F. graminearum in crop residues, the cool, wet weather and the lack of resistant wheat cultivars and efficient chemical control created the ideal environment for a devastating FHB epidemic. Fusarium graminearum poses a double threat to wheat as it decreases grain yield and produces trichothecene mycotoxins, such as deoxynivalenol, which reduce quality of the grain. Deoxynivalenol (DON), also known colloquially as vomitoxin, is the most commonly encountered Fusarium toxin in food and feed (36). Deoxynivalenol was isolated and described for the first time in 1973 and is often associated with feed refusal and emesis in swine (1, 177) (Figure 1-2). The levels of the DON were as high as 44 parts per million (ppm) during the Northern Plains epidemic (106). The FDA and Center for Food Safety and Applied Nutrition issued levels of concern for DON, a mycotoxin which may be found in F. graminearum-contaminated grain. The guidelines
18 5 set forth by the Food and Cosmetics Compliance Program suggest a limit of 2 µg/g (2 ppm) DON in raw grain and 1 ppm in finished flour products intended for human consumption. Grain earmarked for animal feed may contain up to 5 ppm DON, however price reductions apply for lower quality grain. The Brewing and Malting Barley Research Institute imposes stricter standards, limiting DON to less than 0.5 ppm in malting barley, as it causes explosive gushing of the malt and beer. It is difficult to predict DON levels in the malt when barley grain with less than 1 ppm DON is used (157). As a result, many malting companies have zero tolerance policy when it comes to DON. Figure 1-2. The chemical structure of deoxynivalenol (DON, vomitoxin), a trichothecene mycotoxin produced by Fusarium species. Nganje et al. (2004) studied the economic impacts of FHB in the Northern and Central Plains for a three year period (1998 to 2000). Direct economic effects from FHB disease are reflected in the loss in production and reductions in price of wheat. Secondary economic effects from an FHB epidemic are felt off the farm, in the regional economy. Secondary effects include any activity and expense associated with crop production with or without disease, such as public utilities, household expenses, transportation, real estate, insurance and local business. The total economic impact from FHB for these three years was almost $2.7 billion; $871 and $1,809 million for direct and secondary effects, respectively (117).
19 6 Biology of Fusarium graminearum The primary causal agent of FHB epidemics in the United States is Fusarium graminearum Schwabe (telomorph: Gibberella zeae (Schwein) Petch). In a field study in North Dakota, F. graminearum was the most common species of Fusarium collected from wheat heads exposed to field conditions for a 24 hour period (103). Fusarium graminearum infects via the floral tissues and can result in characteristic blighted symptoms on barley, oats and rice, as well as wheat (59). In addition to causing head blight, F. graminearum is one of the causal agents of stalk and ear rot of corn. Corn is also at risk of deoxynivalenol contamination. This species has also been shown to infect soybean seed and seedlings in Ohio (25). Many other crops experience colonization by this cosmopolitan fungus, making it difficult to interrupt its life cycle through crop rotation. To study the genealogy of F. graminearum six nuclear genes were sequenced from strains representing the global diversity of this pathogen (118). Seven phylogenetic lineages were elucidated and these were also shown to be biogeographically related. This suggests that the lineages may be evolutionarily distinct species, as gene flow has been limited between these genealogies. Lineage 7 includes F. graminearum isolates most commonly associated FHB of wheat worldwide and are able to produce type B trichothecenes. Trichothecenes are divided into two groups (A and B) based on relative toxicity: Type A (e.g. T-2 toxin) are more toxic to humans and animals than type B (e.g. DON, nivalenol) (36). O Donnell et al. (2004) described the evolutionary history of this clade. The Fg clade is composed of nine cryptic species based on mating type locus and eleven nuclear genes (119). The members of the Fg clade were found to possess both mating types and are therefore homothallic. Based on this information, it was determined
20 7 that these distinct homothallic species had evolved from a single ancestor. Goswami and Kistler (2005) examined the aggressiveness and toxin production of strains within eight of the nine cryptic species. The strains were isolated from various substrates, including non-gramineous hosts. According to point inoculation pathogenicity tests, members of all 8 species were able to cause disease on wheat and spread throughout the wheat spike. All strains produced trichothecenes in wheat. Therefore, all cryptic species tested within Lineage 7 were pathogenic and virulent. However, aggressiveness and the amount of toxin produced were strain-dependent rather than species-dependent (60). In this study, highly aggressive isolates were defined as those which caused disease severity in wheat that was not significantly different from that of the positive control strain (100% severity). This study concluded that amount of trichothecenes produced, rather than the type, was a major factor for FHB on wheat (60). Trichothecene production profiles (chemotypes) for the Fg clade do not correlate well within lineages, determined by six nuclear genes (181). A chemotype is a chemical phenotype, that is the profile of natural compounds, including mycotoxins, an organism produces (37). The trichothecenes associated with FHB epidemics are deoxynivalenol (DON), 3-acetyldeoxynivalenol (3-ADON), 15-acetyldeoxynivalenol (15-ADON) and nivalenol (NIV). Three chemotypes were identified within the type-b trichothecene lineage: the NIV, 3-ADON and 15-ADON chemotypes (181). The NIV chemotype produces predominately nivalenol, fusarenon-x and small amounts of DON. The 3- ADON chemotype produces DON and a greater proportion of 3-acetyldeoxynivalenol than 15-acetyldeoxynivalenol. Finally, the 15-ADON chemotype also produces DON and a greater proportion of 15-acetyldeoxynivalenol than 3-acetyldeoxynivalenol. The 3- and 15-ADON chemotypes do not produce nivalenol. Sequencing a region of the trichothecene biosynthesis cluster suggested that an ancestor of the Fg clade possessed all
21 8 of the trichothecene polymorphisms currently detected. Chemotype polymorphism observed among and within lineages today has been suggested to be the result of balancing selection in Fusarium populations (181). Today, a strain of F. graminearum can be chemotyped by analyzing chemotype-specific polymorphisms present the trichothecene biosynthetic gene cluster (37, 85, 180). Fusarium graminearum may survive from season to season on crop residues as a facultative saprophyte (120). Perithecia, the sexual reproductive structure, are produced on crop stubble during favorable environmental conditions. Dufault et al. (2006) found Gibberella zeae, inoculated on corn stalk pieces with high water potential (-0.45 MPa), produced mature perithecia in 10 days at temperatures of 20 and 24 o C. Stubble with water potential of MPa produced perithecia after 10 days at 20 o C but took 15 days at 24 o C. This study also found a greater number and size of perithecia to be produced between 16 and 24 o C after 20 days. Temperatures below 10 o C or above 28 o C and low moisture levels limited perithecia formation and maturation (44). Mature ascospores are forcibly discharged from perithecia into the air and then dispersed by wind. Schmale et al. (2006) found the airborne populations of G. zeae to be quite diverse. Ascospores may originate from multiple geographic locations and are distributed over great distances (153). Francl et al. (1999) found the daily median inoculum level of G. zeae on wheat spikes to be 20 colony forming units in an epidemic field. In areas experiencing moderate to severe epidemics more than 50 colony forming units could be detected on wheat spikes daily, suggesting that multiple infections contribute to these epidemics (51). Macroconidia, the asexual spores, are produced on sporodochia and rely on splash dispersal. The macroconidia of F. culmorum and F. graminearum, are multi-cellular and slightly sickle shaped. Horberg (2002) studied the splash dispersal patterns of Fusarium conidia and found spore morphology to have no
22 9 apparent effect on dispersal. Macroconidia reached a vertical height of 58 cm from the inoculated straw substrate. Inoculum reached a distance of 100 cm and new colonies were formed from individual conidia as well as aggregates (67). Trichothecene Mycotoxins As mentioned previously, F. graminearum contaminates grain with mycotoxins. Mycotoxins are secondary metabolites of fungi that are harmful to humans and animals at low concentrations (102). Fusarium strains which cause FHB are capable of producing zearalenone, an estrogenic toxin more commonly found in corn than wheat, and trichothecenes (NIV, DON and ADONs). DON is a sesquiterpene epoxide, a tricyclic molecule with a highly reactive epoxide group. The biosynthesis of DON begins with farnesyl pyrophosphate which is catalyzed into the cyclical trichodiene by trichodiene synthase (38). Trichodiene then undergoes a series of chemical reactions to form DON, NIV, 3-ADON and 15-ADON from a single pathway (38). Genes controlling trichothecene biosynthesis, including Tri5 which encodes the trichodiene synthase, have been characterized and the majority are located in a gene cluster (80). Deoxynivalenol inhibits protein synthesis by physically binding to eukaryotic ribosomes, making this trichothecene toxic to plants, animals and humans (73). Specifically, DON blocks translation by interfering with the peptidyl transferase (172). DON is acutely phytotoxic as it retards plant growth, reduces seedling germination and causes necrosis (96, 145, 179). In fact, wheat spikelets inoculated with DON produce the characteristic bleaching symptoms within 5 to seven days (96). Symptoms spread throughout the wheat head and kernel set was inhibited above the DON-inoculated spikelet. Deoxynivalenol is water soluble and precedes fungal growth through the vascular tissue (164). The toxin has been detected in wheat kernels that appeared healthy
23 10 upon visual inspection (162). Following wheat head dissection, the highest median concentrations of DON were found in the rachis which supports the theory of vascular transport (162). After reaching the rachis, higher levels of DON were found below the point of inoculation, than in spikelets above said point (149). Deoxynivalenol mycotoxicoses are characterized by feed refusal and vomiting in livestock, hence the colloquial name for DON, vomitoxin (102, 177, 178). Swine are most sensitive to DON and ruminants are one of the least sensitive livestock animals. Gastrointestinal disorder in humans has been associated with consumption of wheat products made from moldy grain contaminated with DON (22). This toxin is immunosuppressive in high doses by promoting leukocyte apoptosis (132). Feeding exercise-stressed mice small quantities of dietary DON (2 ppm) for two weeks resulted in immunotoxicity (92). The effects of long-term, low-dose intake of DON have not been studied in humans. However, a urinary biomarker has been developed to study the DON intake of animals and humans (107). Meky et al. (2003) studied the urinary DON levels of two populations living in regions of high and low-risk exposure in China. The daily DON intake in high-risk and low-risk areas was 1.1 to 7.4 and 0.3 to 1.4µg/kg bodyweight, respectively (107). The World Health Organization (2001) issued a tolerable daily intake guideline of 1µg of DON per kilogram bodyweight. While not the most toxic mycotoxin, humans are exposed most frequently to DON than other mycotoxins. Deoxynivalenol biomarkers were found in 98.7% of participants in a United Kingdom study examining the intake of common items, such as bread, cereal and pasta (171). Reducing wheat intake in the diet is associated with reduced DON exposure (170). Many mycotoxins may also play an important role in fungal pathogenesis as virulence factors (38, 39). Mutants of F. graminearum that do not produce DON are less virulent than wild type strains (141). Proctor et al. (1995) demonstrated that the virulence
24 11 of F. graminearum mutants lacking a functional Tri5 gene was significantly reduced in susceptible spring cultivar Wheaton. The virulence of this mutant was also reduced on rye, inconsistent on oats and exhibited wild type virulence on maize. Furthermore, the ability of F. graminearum to spread within inoculated wheat heads is also conferred by the presence of DON (12). Bai et al. (2002) found that a non-toxigenic strain of F. graminearum was unable to colonize wheat spikes beyond the inoculated floret. Jansen et al. (69) studied the effects of wild type and non-toxigenic mutant F. graminearum inoculations of barley and wheat. In the absence of trichothecenes, fungal translocation to the rachis in wheat was inhibited by the development of cell wall thickening at the rachis node. It was hypothesized that this defense response is inhibited by trichothecenes produced by wild type F. graminearum. This study concluded that trichothecenes may not be involved in initial infection, but are important virulence factors for fungal colonization of wheat and barley. Deoxynivalenol is also capable of inducing plant defense response genes. Wheat leaves that were infiltrated with DON showed hydrogen peroxide, a reactive oxygen species (ROS), production within 6 hours of exposure (40). While ROS may aid in antimicrobial defense, they also result in host cell death, providing dead plant tissue that favors growth of the necrotrophic pathogen F. graminearum. Disease Symptoms and Signs Initial F. graminearum infections may result in small water-soaked lesions on the glumes. Soon after, the fungus invades the xylem and phloem tissue of the rachis, potentially inhibiting vascular transport and causing the characteristic premature bleaching symptoms of wheat spikelets (78) (Figure 1-3). Contamination with DON alone can also cause cell death and bleaching of the floral tissue (96). Following
25 12 inoculation during anthesis, FHB symptoms may be visible on the wheat head five to seven days depending on environmental conditions (8). Under humid conditions, pink mycelium may be seen growing on the outside of infected florets. Symptoms of the kernels include slight wrinkling or more pronounced shriveling; severe infections may result in tombstone kernels which are shriveled and white or pink in color (Figure 1-3). The name tombstone is indicative of their resemblance to small pieces of limestone. Severely shriveled kernels exhibit aggressive F. graminearum colonization inside and on the surface of the kernels (16). The shriveled appearance is the result of starch and storage protein degradation by the pathogen (16). Fusarium Head Blight Disease Cycle Fusarium graminearum survives from season to season on crop residue left behind from the previous crop, such as wheat or maize. Under the appropriate temperature and moisture conditions primary inoculum, ascospores and/or macroconidia, is produced. Ascospores are forcibly discharged from perithecia and wind-blown while macroconidia are splash-dispersed onto lower leaves or wheat heads (Figure 1-3). In the U.S., spore production begins in the spring and often coincides with wheat flowering (anthesis). Wheat is considered most susceptible to F. graminearum during anthesis and early grain development. In an inoculum recovery experiment, both ascospores and macroconidia were found during all collection periods during anthesis through grain-fill with a few exceptions (103). One day in 1999, neither spore type was collected and during a series of four days in 2001 only ascospores were observed. Within single wheat heads, the proportion of ascospores varied from 40 to 90%. The average ratio was two ascospores for every one macroconidium. Macroconidia were most often observed during days when overall inoculum density was high. This study concluded that the
26 13 probability of a successful infection is greatest during periods of high inoculum densities and that the presence of both spore types during these periods suggests they are both important inoculum sources. Under optimal conditions (100% relative humidity and 20 o C), F. graminearum macroconidia germinate in as little as 2 hours (21). Generally, hyphae enter through stomata on the glume surface or wounds (139). As hyphae colonize anthers, pollen grains are destroyed which prevents fertilization of other flowers (144). Two days after infection Kang and Buchenauer (2002) found a dense hyphal network had formed on inner surfaces of the lemma, glumes, palea and top of the ovary. The fungus also forms infection hyphae which produce cutinases that break down cell walls and gain access to epidermal cells (78). The photosynthetic ability of the glumes is compromised as hyphae grow throughout chloroplast-containing cells (144). Hyphae also invade xylem and phloem tissue, blocking vascular transport (78). Conidiogenic cells emerge from stomata on glume surfaces and begin to sporulate as early as 48 hours after infection (139). Despite quick colonization of wheat florets and conidial reproduction, FHB is usually considered a monocyclic disease (50). Any secondary inoculum produced has little effect on healthy plants because wheat has a limited window of susceptibility during grain development. Colonization of the wheat head may occur in two ways: (i) horizontally, as the fungus infects contiguous florets of the same spikelet moving towards the rachis, and (ii) vertically in which the fungus accesses the vascular tissue to infect spikelets above and below the point of infection (144). Following point inoculations of a middle floret, greater seed infection was found below the point of infection than above (7). Under the same conditions, the highest levels of F. graminearum were found in the rachis instead of the kernels, also suggesting fungal movement occurs in the vascular tissue.
27 14 The timing of infection is very important for disease development and severity. Hart et al. (1984) indicated that the earlier kernels become infected, the greater the reduction in grain weight. Infections that occur via the anthers prevented the wheat kernel from forming (62). Infected kernels may germinate to produce blighted seedlings which are not likely to reach maturity (56). Following harvest, the remaining wheat residue may harbor inoculum until the next spring, thereby completing the disease cycle (Figure 1-3). Infected kernels usually weigh less than healthy kernels and are often blown from the combine back into the field. These kernels provide an inoculum couse for subsequent crops and have been shown to support a higher level of perithecia production than other wheat residue types (125). Other susceptible hosts, including barley, oats, corn and rye, as well as weed species also allow F. graminearum to overwinter (126).
28 15 Figure 1-3. The schematic demonstrates the disease cycle of Fusarium Head Blight, beginning with inoculum dispersal in the spring and finishing when wheat is harvested in the late summer. Disease and Mycotoxin Management I. Cultural Practices Control measures for FHB aare re primarily cultural and include tillage and crop rotation practices. Conservation or no till practices allow F. graminearum to over-winter on crop stubble from the previous season. Conservation tillage is associated with conditions which increase the nnumber of Fusarium species in the soil (165). (165) Deep tillage decreases the chance of Fusarium infections because there is less inoculum available at the soil surface (29). Use of a moldboard plow resulted in significantly less FHB disease severity and incidence than in chisel chisel-plowed or no-till plots (41).. This study also found
29 16 that yield was 10% greater in the moldboard plowed plots. Furthermore, moldboard plow usage and deep tillage lower the diversity and frequency of Fusarium species isolated from soil (165). Tillage also plays a role in decreasing the availability of atmospheric inoculum (ascospores) at the soil surface, thereby reducing the risk of regional epidemics (153). A higher diversity of Fusarium was observed in plots treated with conservation tillage practices. Ideally competition between diverse Fusarium species and other microbes would keep populations of FHB-inducing F. graminearum in check. However, under ideal environmental conditions and in the presence of a susceptible host, FHB epidemics can occur. Other common cultural control methods include crop rotation. Wheat which follows an alternative Fusarium host, such as barley, rye or corn, is at greater risk for mycotoxin contamination and FHB (45). Wheat planted after soybean experienced 25% less DON contamination, than wheat planted after wheat and 50% less, than wheat after corn (41). Dill-Macky and Jones (2000) observed the greatest amount of disease severity and incidence when wheat followed corn and the least when wheat followed soybean. It is common to use a clean-up (non-host) crop, such as soybean, alfalfa or flax, to reduce the population of F. graminearum in a field. However, recent work has shown that F. graminearum isolates which are highly pathogenic on corn may also be pathogenic on soybean seedlings (25). While crop rotation is an important practice for other agronomic issues, such as soil fertility, it may play less of a role in FHB control than previously thought. Planting date may also influence FHB susceptibility for spring wheat. A Canadian study found that the longer wheat planting was delayed after May 9 the greater the disease incidence and severity (167). Cultural control methods provide control of infield (local) inoculum by disrupting the life cycle of the pathogen (20, 41). However, F.
30 17 graminearum spores are able to travel great distances through the atmosphere (101, 154). Local control alone does not provide sufficient protection during severe epidemic years, especially when significant regional inoculum is present (101, 147, 154, 188). Furthermore, conducive environmental conditions can overcome cultural practices, such as tillage (100). In general, an integrated pest management system is recommended. II. Host Resistance Characteristics Complete resistance to FHB has yet to be identified, but many cultivars possessing moderate resistance have been released and are currently in use today. There are many types of hypothesized resistance to FHB including type I, resistance to initial infection, and type II, resistance to spread of disease within the wheat head (157). A single wheat variety may have two types of resistance, only one type or no resistance, as resistance genes are additive (163). Cultivars may be more or less resistant at different developmental stages and forms of partial resistance are common. Schroeder and Christiansen (1963) were unable to extract any plant chemical with antifungal properties from resistant cultivars, suggesting resistance is a physiological property of the plant. Genetic resistance has been shown to increase the incubation period of the disease (the time it takes first symptoms of disease to appear following infection) (144). Symptoms began to appear on type II cultivar Sumai 3 just as disease reached maximum severity on a susceptible variety. A major QTL for FHB resistance, has been identified on chromosome 3BS in Sumai 3 and its derivatives (15). This QTL, also known as Qjhs.ndsu-3BS, has been named FHB1. Resistance mechanisms may also influence the toxin content in grain. Resistance to trichothecene accumulation is known as type V resistance (157). Type V resistance is divided into two classes (23). Class 1 is characterized by mechanisms which involve the
31 18 degradation or detoxification of trichothecenes. One example of this is the formation of DON-3-glucoside, a conjugated or masked mycotoxin found in naturally contaminated grain alongside DON (18). It is hypothesized that the QTL FHB1 encodes for a DONglycosyltransferase (96, 137). While conjugated DON compounds are less toxic to eukaryotic cells than DON, toxicity is restored by hydrolysis, during digestion (23, 137, 184). Class 2 resistance includes mechanisms through which mycotoxin synthesis is inhibited (23). Plant compounds, such as antioxidants, are known to interfere with trichothecene biosynthesis. Wheat breeders face many research challenges while assessing resistance to FHB. As environment is crucial to disease and toxin development, it is recommended that greenhouse trials be confirmed by field trials when screening germplasm for FHB resistance (13). Greenhouse conditions favor early and fast disease development, whereas field trials are subject to environmental variability. Miedaner et al. (2003) attempted to assess the resistance genotype of wheat cultivars by observing the phenotype produced by two inoculation methods. Spray inoculation assessed type I while point injection of a single floret measured resistance to the spread of disease (109). Both spray and point inoculations resulted in disease and produced similar disease severity on the same cultivars. That study concluded that performing both inoculation methods will provide additional information about host resistance as type I and II are controlled by different loci. However, these theorized types of resistance are often considered an oversimplification (27). III. Fungicides There are conflicting reports on the effectiveness of fungicides to control disease and mycotoxin production. This is likely due to the complex interactions between
32 19 fungicides and target versus non-target organisms, application strategies and environment. It is difficult for growers to predict when fungicide applications will be most effective in disease prevention. Disease incidence is sporadic from year to year and ultimately dependent on weather conditions during key periods in wheat development. Tebuconazole, a triazole fungicide, has been considered one of the most efficacious fungicides against FHB (97). Until recently, tebuconazole fungicides (e.g. Folicur 4F) were only used under emergency exemptions (section 18) during severe epidemic years. During May 2008, the Environmental Protection Agency granted full registration (section 3) to Folicur 4F for FHB control in wheat and barley (64). Simpson et al. (2001) treated naturally-infested wheat plots with combinations of azoxystrobin and tebuconazole fungicides. F. graminearum was not detected in wheat heads of untreated plots but was found in heads which were treated with azoxystrobin, a strobularin (161). This European study also found azoxystrobin to affect DON levels. To examine fungicide efficacy on mycotoxin accumulation the wheat heads were treated with azoxystrobin or tebuconazole following spray inoculations with F. culmorum. Grain from untreated heads contained an average of 3.13 ppm DON, while azoxystrobin and tebuconazole-treated grain contained 5.45 ppm and 1.38 ppm respectively. However there was no increase in the amount of F. culmorum in the azoxystrobin-treated grain. A study by Mesterhazy et al. (2003) uncovered a similar phenomenon. Plots of winter wheat were sprayed with several fungicide treatments during mid-anthesis; spray inoculations with F. graminearum and F. culmorum took place the following day (108). While all fungicide treatments reduced disease severity, kernel damage and yield loss, azoxystrobin treatments showed an increase in DON levels compared to untreated plots. In a controlled greenhouse study, azoxystrobin reduced FHB severity by 30 to 55%, and the fungicide did not directly stimulate DON production (134). It is thought that
33 20 strobilurin fungicides, such as azoxystrobin, effectively eliminate other fungal competitors of Fusarium. Triazole fungicides, such as tebuconazole, prothioconazole and metconazole, are able to reduce disease and mycotoxin contamination, but lose efficacy when applied at low concentrations (108, 134). Two days following exposure to tebuconazole Kang et al. (2001) observed thickened cell walls, increased septation and excessive hyphal branching in F. culmorum. The exposed colonies did not show significant growth after three days of exposure and the cytoplasm became necrotic. Deoxynivalenol production was reduced in fungicide-treated hyphae (79). Uniform fungicide trials have been conducted to assess fungicides and application strategies across different wheat growing regions. A meta-analysis examined the effects of tebuconazole, known under the brand name of Folicur, on FHB disease index (a combination of FHB severity and incidence) and DON accumulation (121). Tebuconazole reduced disease index by 40.3% and reduced DON accumulation by 21.6%. Overall, higher levels of disease control and DON reduction were observed for spring wheat than winter wheat. Uniform fungicide trials also showed that the combined effect of prothioconazole and tebuconazole was to reduce FHB index by 52% (122). Used individually, prothioconazole and tebuconazole reduced index by 32 and 40%, respectively. Metconazole, prothioconazole alone and a combination of prothioconazole with tebuconazole also reduced DON by 45, 43 and 42%, respectively. Tebuconazole alone only reduced DON by 23%. This work suggested that other triazole fungicides may be superior to tebuconazole in terms of disease and mycotoxin control (122).
34 21 IV. Role of Fertilizers Cereals subjected to nitrogen fertilizers are at higher risk of FHB (45, 94). Lemmens et al. (2004) observed an increase in disease intensity and DON levels as greater levels of nitrogen were applied. However, other studies have found nitrogen fertilizers to have little effect on disease (167, 189). In a Japanese study, researchers applied nitrogen at anthesis, inoculated shortly thereafter and observed no significant stimulatory effects on disease or mycotoxin levels in the greenhouse nor in the field (189). During unfavorable environmental conditions, over-fertilization has been shown to increase mycotoxin accumulation (63). If appropriate nitrogen concentrations and application timing strategies are followed, the plant-health and yield benefits of fertilizers outweigh the risk of possibly stimulating FHB. V. Biological Control for FHB Management Ongoing research examines the effects beneficial microorganisms or biological products, which may provide some control of FHB in the field. The bacterium Lysobacter enzymogenes strain C3 produces lytic enzymes that are damaging to fungi (72). Jochum et al. (2006) inoculated wheat plants with broth cultures of L. enzymogenes C3 and then inoculated the plants with F. graminearum one week later. Plants which received the biocontrol treatment experienced less than 10% FHB severity compared to controls which had over 80% FHB severity (72). Bacterial cultures were heat-treated to kill the bacterial cells and denature lytic enzymes. The heat-treated cultures were also able to reduce FHB severity, suggesting another mechanism of L. enzymogenes to induce host resistance. Bacillus mojavensis RRC 101, a bacterial endophyte of wheat seeds, may provide protection from Fusarium seedling blight. Seedling emergence of a FHBsusceptible cultivar improved from 20% to 82%, when seeds were co-inoculated with B.
35 22 mojavensis and F. graminearum (11). Other biocontrol agents, such as the fungus Trichoderma harzianum, have been used to decompose wheat stubble in the fields thus preventing inoculum build-up (68). Cryptococcus yeasts were also found to be effective at decreasing disease severity (84). However, FHB management involves controlling disease and mycotoxin accumulation. Psuedomonas florescens and P. frederiksbergensis strains reduced disease severity by 23% and improved wheat and barley disease-associated yield losses by 16% (81). These bacteria were more efficient at reducing FHB when applied 24 hours before Fusarium inoculation, than after pathogen inoculation. P. florescens strains were also able to significantly reduce DON accumulation in grain by over 70% (81). The biochemical chitosan has also been shown to reduce disease and DON in the field (82, 83). Chitosan is derived from crab shells and induces defense responses, such as peroxidase production and lignin deposition, in wheat (175). In a field setting, chitosan reduced FHB symptoms by 76% compared to positive controls (82). Khan and Doohan (2009) also found chitosan treatment to reduce DON accumulation by over 70%. Environmental Effects on Disease and Mycotoxin Production Despite control measures, such as planting moderately resistant cultivars, FHB epidemics are profoundly influenced by the environment. In general, F. graminearuminduced FHB epidemics are associated with warmer and wet conditions (187). Schaafsma et al. (2001) found the majority (48%) of differences in DON contamination are explained by the different weather patterns of each year. They also found that wheat cultivar accounts for 27% of the variation in DON accumulation and crop rotation accounts for 11 to 28% (152). Other studies suggest that the environmental profiles that favor F. graminearum growth and DON accumulation may be slightly different (66).
36 23 Disease forecasting models have become important tools for wheat growers (28, 34, 86, 150). These models estimate the potential risk for FHB based on wheat region, cultivars used and weather patterns and forecasts. By evaluating their risk, growers can make better management decisions. I. Temperature As indicated previously, temperature is influential for fungal growth and inoculum production. Fusarium graminearum reduced yield by nearly 47% when wheat was inoculated and incubated at 20ºC (24). Incubation at 16ºC reduced yield by 33% and these plants also contained less fungal DNA than plants at incubated at 20ºC. However, more FHB symptoms were observed at 16 o C. This suggests small temperature differences may have significant effects on FHB severity and yield. This also indicates that severity and fungal biomass are not always positively correlated. In general, temperatures above 20 o C favor mycotoxin production, especially during early stages of infection (185). Low temperatures ( 10 C) before anthesis (flowering) reduced inoculum production and DON accumulation (65). However, freezing conditions do not have adverse affects on fungal mycelium as normal growth resumed once hyphae thawed (21). This suggests that F. graminearum may produce less DON during cool periods, but is able to survive periods of frost. High temperatures ( 32 C) after anthesis also negatively affect DON production (65). Martins and Martins (2002) found no mycotoxin production to occur at temperatures over 37ºC when F. graminearum was inoculated on cracked corn kernels. This study also found optimal DON production to occur during incubation at 22ºC (104). Hope et al. (2005) found DON accumulation to peak after 40 days at 25ºC when wheat grain was inoculated with F. graminearum.
37 24 II. Moisture and Humidity Rainfall is required for the production and maturation of perithecia which produce the main source of primary inoculum. Increased disease severity was observed when inoculations during anthesis were accompanied by a three day wet period (90). Following late milk/early dough seed development stages, irrigation period duration had no effect on subsequent disease severity. Rain either facilitated colonization by F. graminearum from an earlier infection or favored infections of maturing wheat heads in the canopy (65). However, a 24 hour inoculum recovery study did not find a correlation between rainfall and levels G. zeae inoculum collected from wheat spikes (103). This suggests that moisture within a 24 hour window may not have an immediate effect on the dispersal or presence of ascospores and/or macroconidia on wheat heads. Moisture may play a greater role in the development and maturation of spore-producing structures and the spores themselves, as discussed previously. During a season when rain fell in greater volume and frequency twelve days preand post-heading, Hooker et al. (2002) found DON concentrations greater than 1 ppm in 94% of fields surveyed and greater than 5 ppm in 60% of fields. In three other seasons, without this rainfall, DON levels remained below 1 ppm 50% of the time in the field. Simulated wet seasons, created by irrigating experimental plots, resulted in lower levels of DON (33, 93). As disease pressure increased, DON decreased and it was thought that premature senescence caused by fungal constriction of vascular tissue prevented the transport of DON (93). Generally, FHB is favored by high moisture, but rainfall may also decrease the availability of inoculum or result in run-off of water soluble DON (93).
38 25 The timing of moisture events relative to wheat head development affects DON accumulation. Wheat is thought to be most susceptible to FHB during anthesis. However, studies have found rainfall to influence disease and DON throughout kernel development. Hooker et al. (2002) found that DON accumulation was influenced by rainfall seven to ten days after anthesis. Cowger et al. (2009) found that 10 and 20 days of post-anthesis moisture to significantly increase disease incidence and severity, compared to wheat that received no post-anthesis misting (30). Moisture has also been shown to increase the amount of DON in fully ripe kernels, when measurements were taken before and after a rain event (151). Infection Characteristics within Wheat Heads Infection patterns differ between susceptible and resistant genotypes. In resistant cultivars F. graminearum colonization was limited to areas of the wheat head near the point of inoculation (7). In susceptible cultivars, the pathogen was more likely to colonize spikelets below the point of inoculation (7). In theory, type II resistance limits the spread of disease. However, due to the role of DON in Fusarium pathogenesis, resistance to FHB is likely more complicated than previously thought (27). Recent work concerning the mechanisms and patterns of fungal infection and subsequent mycotoxin accumulation in wheat heads suggest DON may be translocated through the vascular system to reach wheat head tissues not colonized by fungus. Following a point inoculation of a single floret, DON was found in xylem vessels and phloem sieve tubes in areas free of Fusarium (75). Toxins were translocated upward by xylem and phloem with transduction downward taking place via phloem only. This study also found that DON was produced in hyphae prior to penetration of the glumes, and the highest concentration of toxin was found in plant cells in closest proximity to the hyphae.
39 26 Furthermore, competition between co-inoculated strains of F. graminearum may lead to increased DON production and less fungal biomass (186). Snijders and Krechting (1992) studied the relationship between DON concentration and ergosterol, a component of fungal cell membranes, in the chaff and kernel. Four weeks after inoculation with F. culmorum at anthesis, levels of ergosterol were low to non-detectable in the kernels of both resistant and susceptible lines. DON levels were relatively high in both the resistant and susceptible kernels (20 ppm and 95 ppm, respectively) despite the lack of fungal biomass. This indicates that little fungal growth is required for toxin production. Eight weeks after inoculation, ergosterol in kernels of the resistant and susceptible lines increased from non-detectable to an average of 12 ppm and 54 ppm, respectively. Mean DON concentration decreased from 95 ppm to 63 ppm over the four week period in the susceptible line (164). This is consistent with the observation that DON levels declined as kernels reach physiological maturity (157). This study supports the concept that fungal biomass is not necessarily indicative of mycotoxin concentration. Conditions which support fungal growth are not necessarily the same as those which stimulate DON production and translocation. Summary The relationship between Fusarium Head Blight disease intensity and mycotoxin accumulation has been extensively studied in recent years. However, there are still many facets of this disease that are not fully understood, especially mycotoxin accumulation during early stages of infection and mycotoxin production relative to infection-timing. It is generally accepted that moisture stimulates F. graminearum infections of wheat, but the timing of these moisture events is critical. Temperature, in addition to moisture, influences fungal growth and mycotoxin production. Deoxynivalenol production may
40 27 occur in temperatures that inhibit fungal growth. These conditions, along with host genotype, may influence the relationship between FHB symptoms and toxin accumulation in grain. Deoxynivalenol translocation within wheat heads has been documented, but the role of environment and host on translocation has not been examined. Fusarium Head Blight and DON are reoccurring problems for the wheat industry due the conservation tillage movement and the lack of completely resistant wheat cultivars, amongst other reasons. Therefore, research must address these issues regarding disease development and mycotoxin accumulation in order to better understand this disease and provide management options. Thesis Hypotheses 1. Infections during anthesis result in moderate disease intensity and substantial levels of DON. 2. Infections during the grain-filling stages of kernel development result in low disease intensity yet significant levels of mycotoxins (> 2 ppm). 3. Infections during both anthesis and grain-fill have additive effects on disease and DON. 4. Warm temperature (22ºC) following inoculation promotes fungal colonization of wheat heads and DON production. 5. Cool temperature (15ºC) following inoculation limits fungal growth but allows for DON production and translocation. Objectives for Research 1. To characterize the relationships between disease intensity (incidence, severity and kernel damage) and deoxynivalenol accumulation with respect to infection-timing.
41 28 2. To characterize the relationships between disease intensity (incidence, severity and kernel damage) and deoxynivalenol accumulation with respect to host genotype. 3. To develop and modify protocols for extracting trichothecenes and ergosterol from small pieces of wheat tissue, such as single florets. 4. To develop a gas chromatography-electron capture detector method to detect fungal biomass and trichothecenes within a single wheat floret. 5. To determine the effects of temperature on F. graminearum growth and deoxynivalenol production and translocation within wheat heads. 6. To determine the effects of host resistance on F. graminearum and deoxynivalenol production and translocation within wheat heads.
42 29 Chapter 2 EFFECTS OF I FECTIO -TIMI G DURI G WHEAT DEVELOPME T O FUSARIUM HEAD BLIGHT A D DEOXY IVALE OL ACCUMULATIO Introduction Fusarium Head Blight (FHB) is an important disease of small grains, including wheat (Triticum aestivum L.). In the Upper Midwest region of the United States, total losses can exceed $1 billion in a single year (106). Epidemics continue to occur throughout the wheat growing regions and are highly dependent on environment (31, 117). In the U.S., the primary causal agent of FHB is the fungus Fusarium graminearum Schwabe (telomorph: Gibberella zeae (Schwein) Petch) (25). Fusarium graminearum over-winters in temperate zones on crop residues from wheat, barley or corn, that remain in the field following harvest under minimal or no till practices (41). These residues serve as a source of inoculum, including ascospores and macroconidia, during the following growing season. Infections occur via the floral tissues of wheat heads and dense fungal hyphae form on the glumes, lemma, palea and ovary as early as two days following contact with host tissue (78). The fungus also invades the xylem and phloem tissue of the rachis, colonization and proliferation within these tissues may inhibit vascular transport of water and photosynthates (78).
43 30 Fusarium Head Blight results in yield reductions and the development of shriveled, tombstone kernels, indicating starch and protein degradation (16, 106). Fusarium graminearum also decreases grain quality by contaminating the grain with trichothecene mycotoxins, predominately deoxynivalenol (DON). This compound is phytotoxic, as DON causes the typical bleaching symptoms associated with FHB without presence of the fungus (96). Deoxynivalenol has been shown to be a virulence factor for F. graminearum pathogenesis (141). Mutants, incapable of synthesizing trichothecenes, exhibited normal growth yet were less virulent than parental and revertant strains when inoculated onto wheat heads in the field (39, 140). It has also been suggested that DON, a water-soluble compound, is secreted by F. graminearum and precedes fungal colonization within wheat heads vascular tissue (164). Following inoculations of a central spikelet, DON was detected in spikelets within the same wheat head uncolonized by fungal hyphae (75). Therefore, the losses resulting from FHB epidemics are two-fold: yield reductions and toxin contamination. Deoxynivalenol binds specifically to eukaryotic ribosomes, resulting in the inhibition of general protein synthesis and cell death (73). Deoxynivalenol, also known as vomitoxin, mycotoxicoses are characterized by feed refusal and vomiting in livestock. While the effects of long-term, low-dose intake of DON on humans are not known; a diet of 2 µg/g (2 ppm) DON for two weeks indicated immune system suppression in exercisestressed mice (92). In the interests of human health, the Food and Drug Administration has issued guidelines limiting DON to 2 ppm in raw grain and 1 ppm in finished flour products intended for human consumption. It is critical for grain millers to adhere to these guidelines, and as a result, contaminated grain receives a lower price. Many studies have illustrated the importance of moisture for FHB infections, disease progression and DON accumulation in wheat (33, 90). The level of inoculum on
44 31 wheat heads has been shown to increase during wet periods (51). Not only does rainfall cause dispersal of F. graminearum spores, specifically macroconidia, moisture promotes perithecia production and ascospore development (44). High humidity, which may follow a rain event, promotes the germination of ascospores (57). Early work by Atanasoff (1920) stressed the importance of environmental conditions, specifically rainy and cloudy weather, for symptom development (10). Humidity and moisture at the time of inoculation also contribute to disease severity and yield loss (93). Schroeder and Christiansen (1963) provided wheat heads with 48 hours of humidity post-inoculation and provided additional 24 hours of humidity at weekly intervals until wheat heads reached physiological maturity (157). These weekly periods of humidity resulted in a slight increase of bleached spikelets and a more pronounced increase in the number of infected seeds, than was observed in wheat heads that did not received supplemental humidity throughout kernel development. In contrast, wheat heads which were spray inoculated and incubated in dry conditions showed significantly less disease severity and spikelet damage than wheat heads which were incubated in a humid environment postinoculation (6). In recent years, moisture has been used to predict infection and disease intensity in FHB forecasting models, as it is an essential factor for disease development (34, 65). While the amount of moisture is important to facilitate infections, the timing of these wet periods during wheat development is also critically important for determining disease severity. Historically, wheat was considered most susceptible during anthesis (Zadoks 65, See Appendix) (6, 9, 90, 120). Severe FHB epidemics have been associated with rainfall events during this time period (106). In general, greater yield losses are associated with infections which occur earlier in grain development than those which occur later (35, 62). Infection timing may also affect grain quality. Seeds, from heads
45 32 infected during anthesis, have been described as small, shrunken (less than two-thirds of normal size) and of low weight, while seed from heads infected two to three weeks postanthesis is typically of normal size, although slightly distorted (10). However, postanthesis moisture can result in severe FHB epidemics, as observed in southeastern U.S. in 2003 (31). In a greenhouse study, inoculations at early dough and a subsequent two day moisture treatment resulted in approximately 72% kernel damage and 96% kernel infection (35). While FHB is a monocyclic disease, the period during grain development in which primary infections occur may be wider than previously thought and is especially dependent on the timing moisture events. The effects of moisture and infection timing on DON production and accumulation are less understood and, at times, contradictory. Llorens et al. (2004) found water activity to have no significant effect on trichothecene biosynthesis, when F. graminearum was grown in culture (99). In the field, Hart et al. (1984) demonstrated that DON production depended on hours of head wetness and that host growth stage was not necessarily a factor (62). Subsequent studies have shown that interactions between amount of moisture and infection timing are significant factors affecting DON accumulation and loss. In the field, moisture during early grain development, such as increased rainfall seven days before to ten days after the heading period, has been positively correlated with greater DON levels at harvest (65). In a greenhouse study, inoculations of a highly susceptible cultivar during the watery-ripe stage (Zadoks 71), followed by 48 hours of moisture, resulted in 98 ppm of DON (35). Toxin levels in harvested grain decreased when wheat was inoculated during subsequent stages of development. Culler et al. (2007) found that extended periods of moisture reduced DON levels when applied from inoculation at anthesis until harvest (33). Another simulated wet season reduced DON contamination yet increased disease severity compared to
46 33 wheat that did not receive supplemental misting post-inoculation (93). While FHB severity, kernel-damage and DON are generally correlated (123), the timing of infections and moisture events may affect disease development and DON production differently. In addition to moisture and timing, another factor contributing to FHB development and DON accumulation, is wheat cultivar. While there is no known immunity or complete resistance to FHB, wheat genotype affects susceptibility and DON accumulation (10, 13, 163, 182). Genotypic variation not only confers a host s degree of susceptibility but also when infections may occur. Early research by Pugh et al. (1933) demonstrated that infections could occur in Marquis as early as the boot stage. However, no infections occurred in Prelude during the boot or head emergence stages (142). Infections were observed in Prelude when inoculated at heading and anthesis stages, but disease severity remained lower than that observed in Marquis (142). A study of seven cultivars demonstrated that certain varieties were more susceptible when inoculated at flowering while others showed more infected and damaged kernels when inoculated at soft dough (157). Other cultivars were equally susceptible at all growth stages examined: anthesis, milk and soft dough. For some wheat cultivars, the degree of susceptibility may change over time. Del Ponte et al. (2007) found Norm, a susceptible hard red spring wheat, to be susceptible to F. graminearum infections and DON accumulation from anthesis through hard dough (Zadoks 85) stages (35). Yet Norm was most susceptible to DON accumulation during the watery-ripe stage and most susceptible to kernel infections from anthesis to late milk. A better understanding of the environmental factors that promote symptom development and DON accumulation and when they occur will aid in plant breeding, as well as management decisions on the farm and at the grain mill.
47 34 The objective of this study was to characterize the effects of infections, facilitated by supplemental moisture, during the anthesis and grain-fill stages of grain development. Three soft red winter wheat cultivars with varying susceptibility to FHB were used in this field study to study the effects of host, in combination with infection-timing, on disease incidence, severity, kernel damage and DON. MATERIALS A D METHODS Field Design and Treatment Description A field experiment was conducted during the 2006 and 2007 growing seasons at the Russell E. Larson Agricultural Research Center (Pennsylvania Furnace, PA 16865). Two cultivars with moderate resistance to FHB, Valor and Truman, and one susceptible cultivar, Hopewell, were used in this study. In the fall of 2005 and 2006, the three soft red winter wheat cultivars were planted in a split-plot design, with two replications. Infection-timing treatment served as the main plot factor while cultivar was designated as the subplot factor. Each of the three cultivars was subjected to four treatments for a total of twelve cultivar-treatment subplots per replication. The subplots were 7.43 m 2 each and were separated by a border of Freedom, a moderately resistant soft red winter wheat cultivar (Figure 2-1). All plots were treated with herbicides to control weeds after green-up in the spring but before stem elongation (thifensulfuron and tribenuron, Harmony Extra 75DF, L/km 2 ; Induce, L/km 2 ).
48 35 Figure 2-1. The schematic above details the field design for this infection-timing experiment. One replication consisted of 4 treatment plots (wet-dry, dry-wet, subplots (Hop = Hopewell, Tru = Truman, Val = wet-wet, ambient) each with 3 cultivar Valor ). Inoculations of Field Plots Inoculations took place during mid-anthesis (Zadoks 65) and the late milk (Zadoks 77) stages of grain development. A mixture of four F. graminearum isolates (R , R-06979, R and R-09731; Fusarium Research Center, The Pennsylvania State University, University ity Park, PA) were grown on mung bean agar for 7 to 10 days at 25ºC, under a 12 h diurnal cycle with white and UV lights (49). The fungal isolates used in this study were isolated from Pennsylvanian fields and belonged to the phylogenetically distinct, trichothecene-producing lineage 7 within the F. graminearum clade (119). Macroconidia ia were harvested by scraping the agar plates with a sterile
49 36 microscope slide and filtering through sterile cheesecloth. Using a hemacytometer, the inoculum was diluted with distilled water to a final concentration of 10 4 macroconidia/ml. A surfactant, Tween 20 (polyoxyethylene sorbitan monolaurate; OmniPur, EM Science; Gibbstown, NJ), was added just prior to inoculations (5ml per 8 liters of inoculum) to allow for more uniform coverage of head tissue. A CO 2 -powered backpack sprayer (Model T, R&D Sprayer; Opelousas, LA) was used to spray inoculate the field plots. The sprayer was calibrated to spray at a rate of L/m 2 (16 gal/acre), using Teejet Even Flat Spray tips (TeeJet Technologies, Wheaton, IL). All subplots received two passes, in opposite directions, for a total of liter of inoculum per subplot during both mid-anthesis and late milk stages. Inoculations were performed after 4PM, to minimize solar radiation damage of the spores. The border rows between subplots were not inoculated.
50 37 Figure 2-2. The photograph depicts the spray inoculation of Fusarium graminearum (10 4 macroconidia/ml) on wheat. All plots were inoculated during anthesis and late-milk stages of wheat head development. Misting Treatments To examine the effects of infection-timing on disease intensity and mycotoxin contamination, four misting treatments were designed. The treatments either supplied or prevented moisture during anthesis and/or late-milk stages. Treatments included: a) ambient field conditions; b) dry during anthesis, misting during late milk (dry-wet); c) misting during anthesis, dry during late milk (wet-dry) and d) misting during both anthesis and late milk (wet-wet). To apply the supplemental misting, wooden-framed misting chambers were placed over the plots (Figure 2-3). The frames were covered in clear plastic to ensure misting was directed to the desired plots, yet plants received adequate sunlight while
51 38 undergoing treatments. The chambers were programmed to mist for five minutes every thirty minutes for twelve hours overnight. During the day, the plastic side flaps were rolled up to allow air flow and keep conditions under the misting chambers as close to ambient as possible. Misting treatments began immediately following inoculations and lasted for four consecutive nights. Once misting treatments were finished, the chambers were removed from the subplots. In order to reduce the impact of rain or dew events on subplots during the dry treatment, two mobile greenhouses were used. The mobile greenhouse consisted of a movable quanset-shaped plastic roof stretched over a metal frame. The frame could be moved back and forth on a track system to cover the experimental plots. The greenhouse was connected to a leaf wetness sensor so that the roof covered the field only during rain or dew events. Thus the plots undergoing wet-dry and dry-wet treatments were able to remain dry as indicated. Plots exposed to the wet-wet treatment and ambient conditions were located adjacent to the moving greenhouses. The greenhouses remained operational until harvest.
52 39 Figure 2-3. The photograph shows the wooden-framed mist chambers which were placed over the cultivar sub-plots to provide misting at anthesis and/or late milk stages. The plastic-covered moveable greenhouse in the background is signaled by a moisture sensor to cover these experimental plots during rain events. The track, on which the greenhouse moves back and forth, is observable in the right-hand side of this photograph. Data Collection and Statistical Analysis During the 2006 season, FHB disease severity was assessed during early and late dough stages. However, there was little change in severity between the two assessments. In 2007, the decision was made to perform severity ratings during early dough (Zadoks 83) only. Plot severity was estimated by sampling twenty wheat heads in five random locations of each subplot. The percentage of a wheat head with bleaching symptoms was visually estimated for each of the one hundred heads. Disease incidence per subplot was also calculated by determining the percentage of wheat heads exhibiting FHB symptoms out the one hundred sampled. Weather data, including temperature and rainfall, were
53 40 collected from the Pennsylvania State Climatologist weather station located at the Russell E. Larson Research Center (124). Plots were harvested when the grain reached physiological maturity (July 6, 2006 and July 10, 2007). Grain was harvested and threshed by hand in 2006 to avoid portions of subplots which had lodged. In 2007, lodging was not an issue therefore, grain was harvested mechanically. The harvested grain was rated for the percentage of Fusariumdamaged kernels (FDK), including shriveled and scabby kernels. FDK was measured by placing approximately 10 g of grain in a 60 mm diameter petri dish and estimating the percentage of affected kernels using a visual scale (Figure 2-4) (46). Total FDK for each subplot was estimated by assessing ten 10 g samples of grain. Figure 2-4. This photographic scale, developed by Engle et al. (1998), was used to estimate the percent of Fusarium-damaged wheat kernels per harvested subplot.
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