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
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
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 split plot 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
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, 0.050 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.
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 oC. 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 oC treatment experienced greater Fusarium incidence than those incubated at 15 oC. 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 oC, 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.
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 ...... 11 Fusarium Head Blight Disease Cycle ...... 12 Disease and Mycotoxin Management ...... 15 I. Cultural Practices ...... 15 II. Host Resistance Characteristics ...... 17 III. Fungicides ...... 18 IV. Role of Fertilizers ...... 21 V. Biological Control for FHB Management ...... 21 Environmental Effects on Disease and Mycotoxin Development ...... 22 I. Temperature ...... 23 II. Moisture and Humidity ...... 24 Infection Characteristics within Wheat Heads ...... 25 Summary ...... 26 Thesis Hypotheses ...... 27 Objectives for Research ...... 27
Chapter 2. EFFECTS OF I FECTIO TIMI G DURI G WHEAT DEVELOPME T O FUSARIUM HEAD BLIGHT A D DEOXY IVALE OL ACCUMULATIO ...... 29 Introduction ...... 29 Materials and Methods ...... 34 Field Design and Treatment Description...... 34 Inoculations of Field Plots...... 35 Misting Treatments ...... 37 Data Collection and Statistical Analysis ...... 39 Results ...... 42 Effects of Year ...... 42 Effects of Fixed Factors ...... 44 Pair wise Comparisons of Cultivar Treatment Interactions ...... 45 I. Disease Severity ...... 45 II. Fusarium Damaged Kernels ...... 47 III. Deoxynivalenol ...... 47 Discussion ...... 50
vii Chapter 3. A EW METHOD TO DETECT DEOXY IVALE OL, DEOXY IVALE OL 3 GLUCOSIDE A D ERGOSTEROL I SI GLE WHEAT FLORETS ...... 55 Introduction ...... 55 Materials and Methods ...... 64 Trichothecene Extraction ...... 64 Ergosterol Extraction ...... 64 Peak Identification and Standard Curves ...... 65 Method Recovery and Hyphal Assessment ...... 66 Sample Derivatization ...... 66 Instrumentation and Analytical Program Settings ...... 67 Results ...... 68 Discussion ...... 74
Chapter 4. DEOXY IVALE OL ACCUMULATIO A D FUSARIUM GRAMI EARUM I FECTIO PATTER S I WHEAT HEADS ...... 78 Introduction ...... 78 Materials and Methods ...... 82 Inoculum Preparation ...... 82 Wheat Production in the Greenhouse ...... 82 Point Inoculations ...... 83 Floret Harvests ...... 86 Sample Preparation and Analysis ...... 86 Statistical Analysis ...... 88 Results ...... 88 Incidence of Fusarium in Wheat Florets ...... 89 Deoxynivalenol Translocation ...... 91 Fungal Biomass Estimated by Ergosterol ...... 94 Deoxynivalenol 3 glucoside Synthesis in Wheat Heads ...... 97 Discussion ...... 99
Chapter 5. SUMMARY A D FUTURE DIRECTIO S ...... 107
LITERATURE CITED ...... 111
APPE DIX: ZADOKS’ SMALL GRAIN GROWTH STAGE SYSTEM ...... 123
viii LIST OF FIGURES Figure 1 1. A map illustrating wheat acreage planted in the United States during the 2007 growing season (Source: USDA NASS; www.nass.usda.gov; accessed 2 April 2009)...... 3 Figure 1 2. The chemical structure of deoxynivalenol (DON, vomitoxin), a trichothecene mycotoxin produced by Fusarium species, shows the toxic, unstable epoxide moiety...... 5 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...... 15 Figure 2 1. The schematic above details the field design for this infection timing 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’)...... 35 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...... 37 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...... 39 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...... 40 Figure 3 1. Wheat is able to detoxify deoxynivalenol by forming deoxynivalenol 3 glucoside using glycosyltransferase enzymes in planta ...... 57
Figure 3 2. The chemical structure of ergosterol, C 28 H44 O, is typical of sterols. The molecule is non polar with the exception of the hydroxyl group at the 3 position...... 59 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...... 70
ix Figure 3 4. A) The GC ECD chromatogram shows peaks corresponding to deoxynivalenol (DON, 21.13 min), the internal standard mirex (36.5 min), deoxynivalenol 3 glucoside (DON Gluc, 37.99 min) and 4) ergosterol (ERG, 52.35 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 ...... 71 Figure 3 5. The standard curves for deoxynivalenol, deoxynivalenol 3 glucoside and ergosterol depict the linear relationships between compound concentration and peak area...... 72 Figure 3 6. The GC ECD chromatogram depicts the mean deoxynivalenol (DON, 21.2 minutes, 0.095ppm) and ergosterol (ERG, 52.7 minutes, 252.3 ppm) content of F. graminearum mycelium grown in potato dextrose broth. Mirex (37.9, 1.0 ng/ l) was used as an internal standard...... 74 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:http://www.castonline.ilstu.edu/ksmick/150/150mflower/150 whspik.JPG)...... 84 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 mid anthesis. C) The entire wheat head is enclosed in a plastic bag to incubate for 48 hours ...... 85 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...... 85 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 oC, harvested 2, 4, 6, 8 and 10 days post inoculation and estimated by gas chromatography electron capture detection...... 93
x 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 oC, harvested 2, 4, 6, 8 and 10 days post inoculation and estimated by gas chromatography electron capture detection...... 96 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 oC, harvested 2, 4, 6, 8 and 10 days post inoculation and estimated by gas chromatography electron capture detection...... 98
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 2007 ...... 44 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...... 46 Table 2 3. Infection timing treatment and cultivar have interactive effects on mean Fusarium damaged kernel ratings (%) during the 2006 and 2007 field seasons ...... 49 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...... 49 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 ...... 73 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 oC and harvest at 2,4,6,8,and 10 days post inoculation...... 90 Table 4 2: Fusarium incidence for each harvested day post inoculation, calculated across all cultivars, temperature treatments and individual florets ...... 90 Table 4 3: The effects of fixed factors on DON accumulation in ‘Alsen’ and ‘Wheaton’ wheat florets incubated at 15 or 22 oC and harvested by 2, 4,6, 8 or 10 days post inoculation ...... 92 Table 4 4: Deoxynivalenol accumulation calculated for each cultivar*temperature interaction, across all harvest days and florets ...... 92 Table 4 5: Deoxynivalenol accumulation for each harvested day post inoculation, calculated across all cultivars, temperature treatments and individual florets ...... 92 Table 4 6: The effects of fixed factors on fungal growth and colonization of ‘Alsen’ and ‘Wheaton’ wheat florets incubated at 15 or 22 oC and harvested by 2, 4,6, 8 or 10 days post inoculation, as estimated by ergosterol...... 95
xii Table 4 7: Ergosterol accumulation for each harvest day, calculated across all cultivars, temperature treatments and individual florets...... 95 Table 4 8: Ergosterol accumulation estimated for each cultivar*temperature interaction, across all harvest days and florets...... 95 Table 4 9: Mean deoxynivalenol 3 glucoside and deoxynivalenol accumulation and the ratio between the two compounds in wheat heads ...... 99
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.
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
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 high yielding 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 oC (26).
3
Figure 1 1. A map illustrating wheat acreage planted in the United States during the 2007 growing season (Source: USDA NASS; www.nass.usda.gov; 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
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
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).
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
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
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 oC. Stubble with water potential of 1.03 MPa produced perithecia after 10 days at 20 oC but took 15 days at 24 oC. This study also found a greater number and size of perithecia to be produced between 16 and 24 oC after 20 days. Temperatures below 10 oC or above 28 oC 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
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
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 1.4 g/kg bodyweight, respectively (107). The World Health Organization (2001) issued a tolerable daily intake guideline of 1 g 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
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
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
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 oC), 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.
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).
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 a 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 n umber of Fusarium species in the soil (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 plowed or no till plots (41) . This study also found
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 in field (local) inoculum by disrupting the life cycle of the pathogen (20, 41). However, F.
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
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 DON glycosyltransferase (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 over simplification (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
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
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).
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 FHB susceptible cultivar improved from 20% to 82%, when seeds were co inoculated with B.
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. graminearum induced 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).
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 oC. 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 oC 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 .
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 pre and 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).
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.
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
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.
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.
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).
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 exercise stressed 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
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 post inoculation (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
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 post anthesis is typically of normal size, although slightly distorted (10). However, post anthesis 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
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.
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, 0.0438L/km 2; Induce, 0.1169L/km 2).
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, wet wet, ambient) each with 3 cultivar subplots (Hop = ‘Hopewell’, Tru = ‘Truman ’, Val = ‘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
07661, R 06979, R 07088 and R 09731; Fusarium Research Center, The Pennsylvania
State University, University 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 were harvested by scraping the agar plates with a sterile
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 0.015 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 0.667 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.
37
Figure 2 2. The photograph depicts the spray inoculation of Fusarium graminearum (10 4 macroconidi a/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) ambi ent field conditions; b ) dry during anthesis, mistin g 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 mis ting 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
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.
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
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 Fusarium damaged 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.
41 Two 25 g subsamples of ground grain from each subplot were analyzed for DON concentration. Due to lodging issues in 2006, a Stein mill (Fred Stein Laboratories Inc.,
Atchison, KS) was employed to grind smaller amounts of grain. In the 2007 season a
Romermill (Romer Labs Inc., Union, MO) was used to grind the harvested grain. The
Romermill had a larger grinding capacity than the Stein mill and was better suited to process a larger volume of kernels. During both seasons, DON was extracted from the 25 g subsamples by shaking for 2 hours at 310 rpm with 100 ml of 84/16 (v/v) acetonitrile water. Ten milliliters of the extract were filtered through a clean up column consisting of
0.5 g of a charcoal alumina mix firmly packed between two polypropylene frits. The solid phase of the clean up columns contained 46.6% charcoal (Darco G 60, EM Science,
Gibbstown, NJ) 33.4% aluminum oxide (J.T. Baker, Phillipsburg, NJ) and 20% celite
(EM Science, Gibbstown, NJ). Cleaned extracts were dried under a warm stream of nitrogen gas. Each subsample was then re suspended in 200 l of 80:20 methanol water and transferred to a corresponding 1.5 ml glass vial.
High pressure liquid chromatography (HPLC) with diode array detection (Agilent
1100 Series, Agilent Technologies; Santa Clara, CA) was used to quantify DON in each subsample. A C18 analytical column (Zorbax SB C18, 2.1 x 250 mm, 5 µm; Agilent
Technologies, Santa Clara, CA) and acetonitrile water gradient was used to separate
DON, which eluted around 16 minutes, from the wheat flour matrix. The HPLC program began with 2 minutes of 100% water, followed by 16 minutes of 97% water, 3% acetonitrile and finally 30 seconds of 91% water, 9% acetonitrile. A 100% methanol wash was used to flush the system and clean the column between runs. A known amount of DON standard (Sigma Aldrich, St. Louis, MO) was added to clean wheat samples to create a “spiked” sample. A spiked sample was included in every HPLC analysis to calibrate the analytical software to quantify the concentration of DON in the subsamples.
42 The accuracy of the method was established by analyzing a naturally contaminated sample at a separate facility operated by the United States Wheat and Barley Scab
Initiative. This “check sample” was determined to contain 12 ppm DON by both the outside testing center and our analytical method. The limit of quantification for this method was 0.1 ppm DON.
PROC MIXED of SAS (Version 9.2, SAS Institute; Cary, NC) was used to determine the effects of cultivar and infection timing treatment on disease severity, FDK and DON accumulation. Cultivar and treatment were treated as fixed effects and block
(moving greenhouse) was treated as a random factor. Disease severity and FDK data were transformed using an angular transformation (arcsine √proportion), and DON contamination data were transformed using natural log prior to analyses to homogenize the variance. Models with or without data transformation were compared using Akaike
Information Criterion and log likelihood values for the model fit. Data from 2006 and
2007 were analyzed separately. When there was a significant ( P < 0.05) factor or interaction effect, pair wise comparisons of least square means were made using the
Tukey Kramer adjustment with a family wise error rate of 0.05.
RESULTS
Effects of Year
Disease and DON levels in 2006 were greater than those observed in 2007. In
2006 average disease severity levels per subplot ranged from 0.3 to 24.5%, while in 2007 disease severity ranged from 0.5 to 18.4%. A wider range of disease incidence, the percentage of heads with bleaching symptoms per subplot, was measured in 2006 (3 to
77%) than in 2007 (6 to 48.5%). All experimental plots contained some levels of disease, indicating that the inoculations and misting treatments produced successful infections.
43 The levels of FDK were also greater in 2006 (2.6 to 51.5%) than in 2007 (2 to 28.2%).
Similarly, DON contamination was greater in 2006 (0.5 to 14.3 ppm) than in 2007 (0 to
3.9ppm). Weather conditions varied between the two years during the experimental period, from anthesis to harvest. The average temperature during this time in 2006 was
19.1 oC, the average relative humidity was 88.8% and the total rainfall was 5.23mm. The average daily temperature during wheat head development in 2007 was 16.2 oC, the average RH was 61.9% and the total rainfall was 8.66cm. Overall, the warmer temperatures and higher humidity in 2006 may explain the higher levels of disease and
DON than in 2007. The movable greenhouses and mist chambers protected the plots from ambient rain, so the increased rainfall in 2007 appeared to have little effect on disease and DON. During the anthesis period in 2006, the average temperature was
20.8 oC, and the average relative humidity (RH) was 85.5%. Following the second round of inoculations, during the late milk stages, the ambient field conditions averaged 15.6 oC and 60.2% RH. In 2007, the field conditions during anthesis were 21.1 oC and 65.4% RH.
During the late milk treatment, ambient conditions were 19.3 oC and 69.9% RH.
Inoculations were performed in the late afternoon; however, the anthesis inoculations during 2007 were performed under very hot conditions (28.9 oC). While the treatments were designed to facilitate infections, by supplying moisture and maintaining high humidity, they lasted for a relatively short period of time (4 days at the anthesis and/or late milk stages). Ambient weather conditions certainly had the potential to contribute to overall FHB development. As a result, the field seasons described here represent relatively moderate (2006) and mild (2007) FHB epidemic conditions.
44 Effects of Fixed Factors
Three wheat cultivars, of varying susceptibility to FHB, were used in this study.
All cultivars were infected by F. graminearum and exhibited DON contamination.
Analysis of variance demonstrated that cultivar had significant effects on disease severity in 2006 and 2007 (Table 2 1). Cultivar was also a significant factor for incidence, FDK and DON during both seasons. This indicates that FHB symptoms, observed on wheat heads or kernels, and DON accumulation in harvested grain are cultivar specific responses.
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 2007.
Disease Fusarium- Incidence Disease Severity Damaged Kernels Deoxynivalenol
Effect df a Fb Pr>F c F Pr>F F Pr>F F Pr>F 2006 treatment d 3 66.83 0.0030 5.31 0.1585 267.92 <0.0001 167.46 <0.0001 cultivar e 2 13.56 0.0001 149.53 <0.0001 98.52 <0.0001 19.84 <0.0001 treatment-cultivar 6 2.80 0.0120 15.86 <0.0001 11.00 <0.0001 6.09 0.0002 2007 treatment 3 3.49 0.1662 10.36 0.0234 56.51 <0.0001 61.12 <0.0001 cultivar 2 12.19 <0.0001 65.09 <0.0001 180.66 <0.0001 87.17 <0.0001 treatment-cultivar 6 1.89 0.0839 11.97 <0.0001 14.59 <0.0001 14.41 <0.0001 a Degrees of freedom b F statistic associated with the analysis of variance c Probability associated with the F test. d Four infection timing treatments including wet wet, wet dry, dry wet and ambient conditions. e Three soft red winter wheat cultivars including ‘Hopewell’, ‘Truman’ and ‘Valor’.
Infection timing during wheat head development was facilitated by four different misting treatments. The factor of treatment was found to have a significant effect on disease severity in 2007, but this was not the case in 2006 (Table 2 1). Infection timing was a significant factor for disease incidence in 2006, but not in 2007. However,
45 treatment had significant effects on FDK and DON during both field seasons. This suggests that grain quality factors, such as FDK and DON accumulation, are influenced by infection timing. The relationship between infection timing and incidence or severity is likely influenced by other environmental and host factors. In general, treatments which facilitated infections during the anthesis period (wet dry and wet wet treatments) resulted in more head and kernel symptoms and higher concentrations of DON.
Pair-wise Comparisons of Cultivar-Treatment Interactions
The interaction between treatment and cultivar was a significant factor for incidence in 2006 only (Table 2 1). The interaction between treatment and cultivar was shown to have significant effects on disease severity, FDK and DON in 2006 and 2007.
While treatment and cultivar alone have effects on these FHB disease parameters, the interaction between these fixed factors allows for comparisons between the cultivar treatment subplots within each field season. Due to use of the movable greenhouses, space was a limiting factor for the experimental design. Two complete repetitions were completed during each year. Despite the limited sets of repetitions, significant differences between cultivar treatment subplots were found using the Tukey Kramer test.
I. Disease Severity
In 2006, the wet dry treatment produced significantly greater disease severity in
‘Hopewell’ than in ‘Truman’ (P < 0.0001) or ‘Valor’ (P < 0.0001) (Table 2 2). This same pattern was observed in 2007, where ‘Hopewell’ exhibited greater severity compared to ‘Truman’ (P < 0.0001) and ‘Valor’ (P < 0.0001). Severity values observed for ‘Truman’ and ‘Valor’ under the wet dry treatment were not statistically different from each other in 2006 or 2007, P = 0.9769 and P = 0.5651, respectively. Treatments which
46 facilitated infections during anthesis (wet dry and wet wet) resulted in greater disease severity ( P < 0.013) for all cultivars than treatments which did not in 2006 (Figure 1). In
2007, only the ‘Hopewell’ wet dry subplot experienced significantly more severity
(18.5%), than dry wet (5.6%, P < 0.0001) or ambient conditions (3.1%, P < 0.0001).
Under mild epidemic conditions, ‘Truman’ and ‘Valor’ experienced fewer head bleaching symptoms, and disease severity was less than 5% across all treatments. There were no significant differences, in disease severity, as a result of infection timing in
‘Truman’ or ‘Valor’ (P > 0.05).
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. Year Cultivar Treatment Wet-Wet Wet-Dry Dry-Wet Ambient 2006 Hopewell 14.53 a* 24.50 a 2.07 a 1.93 a B** A C C Truman 6.76 a 8.79 b 0.49 a 0.32 a A A B B Valor 7.53 a 11.27 b 2.45 a 1.26 a A A B B 2007 Hopewell 8.45 a 18.45 a 5.55 a 3.10 a B A B B Truman 2.30 b 1.75 b 0.49 b 1.25 a A A A A Valor 4.57 ab 4.60 b 1.85 ab 1.75 a A A A A *Capitalized letters, located within rows, describe significant differences ( P ≤ 0.05) amongst treatments within cultivars. ** Lowercase letters, located within columns, describes significant differences ( P ≤ 0.05) amongst cultivars within each treatment.
47 II. Fusarium-Damaged Kernels
The wet wet treatment produced more FDK across all three cultivars, than in other treatments (Table 2 3). This observation was statistically significant in 2006, not
2007. The wet wet treatment was designed to stimulate infections during anthesis and late milk. Two periods of infection, as opposed to one infection period during anthesis
(wet dry) or late milk (dry wet), increased FDK. Despite rather low disease severity of head tissues, the wet wet treatment produced a wide range of FDK in ‘Hopewell’
(51.5%), ‘Valor’ (42.5%) and ‘Truman’ (31.5%) in 2006. The effect of the wet wet treatment was most pronounced in ‘Truman’ and ‘Valor’. There were no significant differences in FDK between the wet dry, dry wet and ambient treatments for ‘Truman’ or
‘Valor’. In fact, FDK was less than 10% for all treatment cultivar subplots in 2006.
‘Hopewell’ exhibited an interesting response to the wet wet treatment during both seasons. Infections and increased fungal activity during anthesis and late milk, lead to increased FDK. This is in contrast to disease severity, which was higher in the wet dry treatment than wet wet. In ‘Hopewell’, it is possible that kernels were more susceptible to fungal damage than the glumes as a result of two infectious time periods.
III. Deoxynivalenol
Deoxynivalenol levels in 2006 ranged from over 14 ppm (‘Hopewell’, wet dry) to
0.5 ppm (‘Truman’, ambient) (Table 2 4). In 2007, several subplots had DON levels that were below the limit of quantification (0 ppm), while the highest DON concentration was found in ‘Hopewell’ subjected to the wet dry treatment (3.9 ppm). In addition to having the highest levels of FHB symptoms, ‘Hopewell’ experienced some of the greatest levels of toxin contamination. Infections of ‘Hopewell’ during anthesis only (wet dry), caused the highest DON accumulation and this interaction was significant in 2007 ( P < 0.0001).
48 High levels of disease severity in ‘Hopewell’ were also caused by anthesis infections, but highest FDK ratings were produced by the wet wet treatment. The disease parameter responses of ‘Hopewell’ to the infection timing treatments were different than those of
‘Truman’ and ‘Valor’. The two moderately resistant cultivars responded in a similar manner to the treatments. One exception to this pattern was observed in response to the dry wet treatment in 2006. ‘Valor’, subjected to infections during late milk only, attained
DON levels of 3.7 ppm. This was surprising, considering the levels of disease severity
(2.5%) and FDK (6.3%) calculated for these subplots in 2006. While infections during grain fill may have had little effect on FHB symptoms in ‘Valor’, they produced DON levels that exceed the FDA’s raw grain guideline of 2 ppm for human consumption. The wet wet treatment resulted in just over 11 ppm in all 3 cultivars in 2006, despite very different levels of FDK in each cultivar. The effect of cultivar appeared to have very little influence on DON accumulation as a result of infections during anthesis and late milk (wet wet), but played a larger role when infections were limited to anthesis (wet dry) or late milk (dry wet).
49 Table 2 3. Infection timing treatment and cultivar have interactive effects on mean Fusarium Damaged Kernel ratings (%) during the 2006 and 2007 field seasons. Year Cultivar Treatment Wet-Wet Wet-Dry Dry-Wet Ambient 2006 Hopewell 51.50 a* 30.25 a 8.75 a 18.00 a A** B D C Truman 31.50 c 4.95 b 2.55 b 8.45 b A B B B Valor 42.50 b 5.75 b 6.30 ab 7.05 b A B B B 2007 Hopewell 28.25 a 26.50 a 11.00 a 8.40 a A A B B Truman 6.85 b 3.30 b 2.50 b 2.00 b A A A A Valor 10.90 b 8.15 b 5.85 ab 3.75 ab A AB AB B *Capitalized letters, located within rows, describe significant differences ( P ≤ 0.05) amongst treatments within cultivars. ** Lowercase letters, located within columns, describes significant differences ( P ≤ 0.05) amongst cultivars within each treatment.
Table 2 4. Infection timing treatment and cultivar have interactive effects on mean deoxynivalenol (ppm) in harvested wheat during the 2006 and 2007 field seasons. Year Cultivar Treatment Wet-Wet Wet-Dry Dry-Wet Ambient 2006 Hopewell 11.22 a* 14.29 a 3.07 ab 1.45 ab A** A B B Truman 11.68 a 5.43 b 1.45 b 0.51 b A B C C Valor 11.07 a 6.76 b 3.68 a 2.38 a A AB BC C 2007 Hopewell 1.61 a 3.90 a 1.00 a 0.10 a B A B C Truman 0.69 b 0.34 b 0.00 b 0.00 a A AB B B Valor 0.71 b 0.51 b 0.00 b 0.00 a A A B B *Capitalized letters, located within rows, describe significant differences ( P ≤ 0.05) amongst treatments within cultivars. ** Lowercase letters, located within columns, describes significant differences ( P ≤ 0.05) amongst cultivars within each treatment.
50 Discussion
Results from this study illustrate the importance of infection timing and host genotype for the development of Fusarium Head Blight symptoms and deoxynivalenol accumulation in wheat. While other studies have also described the importance of host growth stage on FHB, controlled conditions were often used (6, 35). The present study utilized movable greenhouses and misting chambers to facilitate infections in a field setting. The objective of this study was to determine the effects of infection timing during anthesis, grain fill or during both stages. All experimental plots were artificially inoculated immediately prior to the infection timing treatments, whether subplots were misted or kept dry. This ensured the presence of inoculum during both growth stages.
Therefore, one can assume differences between subplots in terms of disease severity, kernel damage and DON accumulation were due to treatment and cultivar, not due to inoculum availability. Yearly weather differences led to a moderate epidemic in 2006 and a mild epidemic in 2007. While moisture and rainfall conditions were manipulated with movable greenhouses and mist chambers, ambient temperature and relative humidity during the infection periods could not be controlled. Heat stress may have been a limiting factor for establishing infections during 2007 and may partly explain why disease levels during that year were lower than those observed in 2006. The cause(s) of lodging in 2006 were not known but, lodging is often indicative of a nitrogen fertility imbalance or severe wind (74, 166). Certain wheat genotypes may also be predisposed to lodging (17). However, since lodging was only observed during 2006 it must have been a random issue not a cultivar trait.
Despite these yearly considerations, similar patterns amongst treatments and cultivars emerged during 2006 and 2007. Effects of cultivar treatment interactions were significant despite the small number of replications, which attests to the strength of the
51 relationships described in this research. In the future, additional cultivars could be subjected to the infection timing treatments. Individual cultivars would likely react differently to the treatments, but cultivars that share parental genotypes or the resistance
QTL FHB1 may respond similarly.
Inoculations and misting treatments during anthesis and/or late milk were designed to facilitate F. graminearum infections. Infections during anthesis resulted in higher levels of disease intensity and DON accumulation during 2006 and 2007 than treatments that did not provide misting during this critical phase. Infections during grain fill alone did not stimulate symptom development any more than ambient field conditions. The ambient treatment was the designed as a control treatment. While ambient plots were inoculated, it represented the effects of natural field conditions without any supplemental misting. The relatively low disease severity and minor kernel damage observed as a result of the ambient treatment corresponded to low levels of DON in the grain. In general, visual assessments of FHB, such as disease severity and FDK, are positively correlated with DON concentration (123). Paul et al. (2005) also showed that FDK, which measures the effects of FHB on the wheat kernels themselves, had the strongest correlation with DON. However, the present study suggests that this correlation is influenced by infection period and cultivar.
Host genotype traits determine a plant’s response to a pathogen and mediate subsequent disease development. Response to infection timing, in terms of severity,
FDK and DON, was cultivar specific. In general, ‘Hopewell’, the most susceptible cultivar, experienced the greatest amount of disease and DON accumulation. When comparing the two moderately resistant cultivars, ‘Truman’ generally experienced fewer symptoms and less DON than ‘Valor’. Infections during grain fill, like those facilitated by the dry wet and wet wet treatments, may have different effects in different cultivars.
52 ‘Truman’ may be less susceptible to F. graminearum and DON throughout development, whereas ‘Valor’ experienced more DON accumulation during grain fill. The dry wet treatment produced few symptoms in the field and minimal kernel damage. In 2006, the dry wet treatment resulted in significantly less FDK (8.7%) in ‘Hopewell’, than was observed as a result of ambient conditions (18%). Despite the low degree of symptoms, grain fill infections resulted in DON levels greater than 2 ppm in ‘Hopewell’ (3.1 ppm) and ‘Valor’ (3.7 ppm). As shown in this study, grain fill stage infections are able to overcome a moderately resistance cultivar and produce significant levels of DON. Grain with substantial levels (> 2ppm) of DON and few FHB symptoms has been reported previously (43, 162). To our knowledge, this is the first study to identify grain fill infections as a possible cause of this phenomenon.
It has been reported that DON levels decrease as the wheat kernels mature (33,
164). The mechanisms for how this DON reduction occurs are unknown. Wheat cultivars may possess the ability to conjugate mycotoxins rendering them non toxic and undetectable by common detection methods (18, 137, 190). Culler at al. (33) found wheat, which received misting for 31 to 32 days following inoculations during anthesis, had less DON than wheat that received 15 to 16 days of misting. Under the extended mist treatment, DON levels were found to decrease with each growth stage. Since DON is water soluble, Culler and colleagues suggested that the free moisture accumulating on the wheat heads may have allowed DON to leach. A lack of correlation between FDK and DON was observed in the wet wet treatment. In 2006, all three varieties had relatively high levels of kernel damage, ranging from 51.5% for ‘Hopewell’, 42.5% for
‘Valor’ and 31.5% for ‘Truman’. Yet the DON levels within the subplots were remarkably similar 11.7, 11.2 and 11.1 ppm for ‘Truman’, ‘Hopewell’ and ‘Valor’, respectively. These results indicate wheat cultivars may respond differently to the timing
53 of moisture and may possess different mechanisms for metabolizing DON. In 2006,
DON levels increased with the additional moisture in these two moderately resistant cultivars. In 2006 and 2007, DON accumulation was reduced in ‘Hopewell’ as a result of the wet wet compared to the wet dry treatment, despite the greater degree of kernel damage. This suggests that DON was either leached from ‘Hopewell’ wheat heads, due to the increased moisture or that ‘Hopewell’ combats mycotoxin accumulation by metabolizing DON into a form not recognized by HPLC analyses (i.e. deoxynivalenol 3 glucoside). The DON conjugating abilities of the cultivars used in this study, as well as those of popular cultivars grown today, are not known. Future work may include performing harvests of wheat heads throughout the season and analyzing for DON and
DON 3 glucoside content. This would confirm host metabolism and determine when this reaction takes place in wheat development.
Fusarium infections, relative to wheat growth stage, have received increased attention in recent years. Del Ponte et al. (2007) performed inoculations in the greenhouse from anthesis to hard dough of a susceptible wheat cultivar. Similar to Hart et al. (1984), Del Ponte and colleagues also found wheat to be susceptible through all growth stages. Both studies, along with the research presented here, suggest infections occurring throughout the grain filling stages contribute to mycotoxin contamination.
Furthermore, Bechtel (1985) found that F. graminearum causes the majority of damage to kernels between two and three weeks post anthesis, when kernel moisture is quite high
(16). During the typical FHB disease cycle, wheat heads are susceptible to Fusarium infections for a few weeks between anthesis and grain maturation, when there is less available moisture in the wheat head to support fungal growth. Infections occurring during anthesis are typically thought to result in the most severe FHB epidemics.
Anthesis may not be the most susceptible stage, but early infections may allow more time
54 for symptom development than infections during later stages. On the other hand, infections during grain fill leave less time for spread and progression of the epidemic.
Delaying the start of an epidemic often leads to reduced disease (58), but the same may not be true for mycotoxin accumulation. Deoxynivalenol may be produced quickly following grain fill infections and the reductions in grain quality may be substantial.
Deoxynivalenol has been found in glume and kernel tissues as soon as 36 hours after inoculation (75). Multiple infection periods, created by the wet wet treatment, stimulated cultivar specific responses to FHB and DON. In the present study, the effects of the multiple infection periods were not additive. Disease severity in the wet wet treatment was equal to or less than severity in the wet dry or dry wet treatments. While infections during anthesis and grain fill caused high levels of kernel damage in all cultivars studied, this did not always correlate to high DON levels. The wet wet treatment, which was designed to facilitate infections during anthesis and late milk, warrants further investigation. Additional infection timing periods could also be studied, such as the dough stages of kernel development.
55
Chapter 3
A EW DETECTIO METHOD FOR DEOXY IVALE OL,
DEOXY IVALE OL 3 GLUCOSIDE A D ERGOSTEROL
I SI GLE WHEAT FLORETS
Introduction
Fusarium graminearum Schwabe (telomorph: Gibberella zeae (Schwein) Petch) is an important fungal plant pathogen and mycotoxin producer. It is the causal agent of
Fusarium Head Blight (FHB) of wheat and barley, as well as Gibberella stalk and ear rot of corn (59). Moldy cereal crops have long been associated with illness in livestock, especially in swine. In 1972, much of the corn harvested in the mid western U.S. was predominately infected with F. graminearum and reports of swine feed refusal and vomiting soon surfaced (177). Extracts from 500g of this infected corn were shown to induce vomiting within 25 minutes. Vesonder et al. (1973) isolated the emetic principle of these extracts, elucidated its chemical structure and named it vomitoxin, due to the symptoms observed in swine. Vomitoxin, more formally known as deoxynivalenol
(DON), was classified as a trichothecene mycotoxin and was shown to cause feed refusal
(anorexia) as well as emesis (178). All livestock and animals are susceptible to DON mycotoxicoses, but swine have been shown to be most susceptible (138). Swine display feed refusal after consuming feed with 1 2ppm DON, whereas cattle and poultry can withstand up to 20ppm (129).
Trichothecenes are small tricyclic sesquiterpene toxins which contain an epoxide at the 12 and 13 carbon positions. Deoxynivalenol is of great concern to small grain
56 farmers, livestock producers, millers and brewers, as it is the most commonly occurring
Fusarium toxin in grain (36, 129). All trichothecenes, including DON, bind to the 60S ribosomal subunit and inhibit protein synthesis in eukaryotic cells (75, 110, 129). This also leads to mitogen activated protein kinase production, which induces cellular apoptosis (130). While acute toxicoses are known to cause organ failure and death, outbreaks are rare. Chronic exposure to low levels of DON is a greater concern to animal and human health. Prolonged exposure to DON results in decreased weight gain, altered nutritional efficiency and immunosuppression (130). The maximum tolerable daily intake (TDI), set forth by the European Union, for humans is 1 g DON per kilogram of bodyweight (48). However, this guideline is often exceeded. Meky et al. (2003) estimated daily DON intake for high and low risk Chinese populations to be 1.9 to 13.0 and 0.6 to 2.5 mg/kg/day, respectively. A study from the Netherlands found that 80% of one year old children exceeded the recommended TDI (133). These studies show that
DON contamination of cereals and chronic consumption of these products is a concern around the world, including both developed and developing countries. The U.S. Food and Drug Administration has issued advisory guidelines limiting DON to 1ppm in finished flour products intended for human consumption, 5ppm in animal feed for swine and 10ppm in feed for ruminants and chickens (173). The European Union adheres to stricter standards by limiting DON to 0.75ppm in cereal flour and 0.50ppm in baked goods and breakfast cereals (48). Cereal based baby foods should contain no more than
0.20ppm DON (48). Many developing countries do not have DON guidelines and have limited means to test or regulate cereals for mycotoxins.
Fusarium graminearum infections and subsequent deoxynivalenol production in cereals are highly dependent on environment. The 1972 epidemic, which led to mycotoxicoses in swine and the discovery of DON, was associated with unusually wet
57 weather in the U.S. Corn Belt (177) . In general, moisture has been shown to stimulate
DON production. In the field, Hart et al. (1984) demonstrated that DON production depended on hours of wheat head wetness (62). In recent years, moisture has been used to predict infection and disease intensity in FHB forecasting models (34, 65) . However,
DON is als o a water soluble toxin. Simulated wet seasons are characterized by extended periods of moisture and result in a decrease of DON in harvested wheat grain (33, 93).
This could be because DON was literally washed from the wheat heads in the field.
Another theory involves the ability of wheat to conjugate DON in vivo (18, 96, 137) .
These DON conjugates, such a s deoxynivalenol 3 glucoside (DON gluc ) (Figure 3 1), may describe some of the DON loss described in the field. DON gluc h as been found in wheat, barley and beer (18, 191) . Other Fusarium mycotoxins, such as zearalenone, also experience conjugation in planta . Zearalenone conjugates have also been detected in wheat and corn (47, 61, 156) . Enzymes, known as glucosyltransferases, conjugate DON and zearalenone to a glucose molecule, rendering the mycotoxin s non toxic (89, 136).
This conjugation provides the plant protection from phytotoxici ty, suggesting conjug ation is a general adaptive defense mechanism (137) . In wheat, DON conjugation has been linked to the FHB1 resistance QTL (96).
Figure 3 1. Wheat is able to detoxify deoxynivalenol by forming de oxynivalenol 3 glucoside (DON gluc, DG) using glycosyltransferase enzymes in planta .
58 The concern over these conjugates is that toxicity is restored once the contaminated grain is ingested (53). Garies et al (1990) fed swine known concentrations of zearalenone and zearalenone glucoside and found the urinary biomarker for zearalenone to increase following digestion, suggesting the cleavage of zearalenone glucoside in vivo . This may pose a potential health risk to livestock and humans, if high levels of conjugated mycotoxins are consumed then un conjugated in the body.
Conjugated toxins may also revert to their toxic form during processing. Young et al.
(1984) studied the effect of milling and processing on wheat flour with a known concentration of DON. DON concentration increased by XX during the production of a yeast doughnut (190). It was hypothesized that the yeast hydrolyzed the glucoside moiety from DON gluc, thereby leading to an increase in overall DON levels. While the
FDA limits DON to 1ppm in flour intended for human consumption (173), very rarely is a finished product, such as a doughnut, tested for DON prior to consumption. Previous studies have indicated that DON gluc accumulation in wheat is quite common. Berthiller et al. (2005) found DON gluc in 100% of naturally infected wheat grain collected from
Austria, Germany and Slovakia. However, the ratio of DON to DON gluc varies greatly in grain samples (18, 148). Despite this apparent health risk, very few published studies have reported information on DON gluc synthesis, accumulation and frequency in wheat, feeds or foods. DON gluc is not currently tested for at grain elevators and mills in the
U.S.
In addition to contaminating wheat grain with trichothecenes, F. graminearum colonizes the kernels, reduces yield and renders seed nonviable. Infected grain is often lower in weight than healthy seed, shriveled and discolored. This is due to fungal invasion and destruction of the seed’s endosperm. Severely damaged wheat kernels may be covered with pink mycelium or have a chalky, white “tombstone” appearance. Pink
59 mycelium may also be observed on infected wheat heads prior to harvest. The diagnostic symptom of FHB is premature bleaching of the wheat head. Bleaching may be isolated to a few spikelets or the entire spike may become bleached. Fusarium infects wheat through the floral tissue and has been shown to spread within heads intercellularly, intracellularly and via the vascular tissu e (77).
Fungal infection of small grains can be quantified using ergosterol, a membrane bound sterol (steroid alcohol) specific to fungal cell membranes (Figure 3 2) (159, 160).
Due its specificity, ergosterol is frequently used as a target for antifungal medications and fungicides, as it is not found in humans or plants. In fungi, ergosterol affects cell membrane fluidity and serves as a signaling molecule. Additionally, ergosterol is a precursor to vitamin D and has been used to fortify foodstuffs for human consumption
(143) . Ergosterol has been used as a fungal biomass indicator to estimate fungal content of water, soils and agricultural commodities (71, 113, 160, 176). In barley, the correlation coefficient between ergosterol and fungal contamination quantified by colony forming units was 0.92 (54) .
Figure 3 2. The chemical structure of ergosterol, C 28 H44 O, is typical of sterols. Ergosterol is specific to fungal cellular membranes.
Despite the serious impact of FHB and trichothecenes on the wheat industry, the relationship between fungal growth and mycotoxin production during infection is not well understood. In general, ergosterol (ERG) is positively correlated with DON content
60 (2, 91). Abramson et al. (1998) found linear correlation coefficients between DON and
ERG in harvested grain to be 0.76 and 0.83 for hard and soft wheat, respectively.
Kernels with a reddish appearance have been found to harbor the greatest amount of
DON and ERG (116). However, other research found a lack of correlation between mycotoxin accumulation, FHB disease severity and ergosterol (55, 162). Sinha and
Savard (1997) found DON in 50% of healthy looking wheat kernels. “Asymptomatic” grain, wheat relatively free of symptoms with levels of DON greater than 2ppm, has been reported in the Eastern U.S. (Mike Pate, personal communication). As mentioned previously, DON is water soluble and is able to travel throughout the wheat heads in the vascular tissue, reaching florets uncolonized by the pathogen. Furthermore, comprehensive research designed to measure correlations between symptoms, ergosterol,
DON and DON gluc has yet to be performed. An analytical method is required to study the relationship between fungal colonization and mycotoxin accumulation in wheat heads and single florets. However, there are few described methods designed to simultaneously detect fungal biomass and mycotoxins within the same piece of tissue.
There are techniques designed to study fungal biomass or mycotoxins in the field and in harvested grain. Chromatography is able to identify ergosterol or trichothecenes individually in a wheat matrix, such as fresh tissue or grain. Chromatographic methods are able to detect ergosterol and trichothecenes at low levels from small sample sizes (87,
127, 128, 158). Capillary columns used with gas chromatography analyses offer “high separation potential” of trichothecenes from various matrices (158). In gas chromatography, the sample is injected into a high temperature injection port and is quickly vaporized. The sample then enters the analytical column which contains a high boiling stationary phase. Compounds in the sample are separated based on column length and their affinity for the stationary phase. As the temperature of the column increases,
61 compounds lose affinity and are eluted from the column as pure components into a detector. Mateo et al. (2001) determined gas chromatography with electron capture detection (GC ECD) to be the preferred analysis method for DON, rather than high pressure liquid chromatography with diode array detection. Briefly, the principle of ECD involves a radioactive isotope ( 63 Ni) contained inside the detector cell. When a carrier gas, such as helium, is introduced its molecules are ionized and electrons are emitted.
The electrons are then drawn to the positive electrode in the detector cell. Compounds with high affinity for electrons (“electron capturing”) compete for these electrons once injected into the ECD cell. Electron capturing compounds reduce the concentration of free electrons in the cell, this reduction and relative amount is reflected as a chromatogram peak representing the presence and concentration of the electron capturing compound.
Preferred extraction solvent for trichothecenes intended for GC ECD analysis is acetonitrile water (169), specifically 84/16 (v/v) (32, 169). Trichothecene extracts are then cleaned by passage through a clean up column. A charcoal alumina column removes interfering materials from grains, foods and feeds, but allows DON and other trichothecenes to elute (146). Fluoroacylation derivatization, with hepta fluorobutyric anhydride (HFBA), allows for increased GC ECD sensitivity for trichothecenes in complex matrices (32, 158). By adding electron combining moieties, such as fluorine (a halogen), to trichothecenes the electron combining ability of these compounds is enhanced. Using these methods, Croteau et al. (1994) achieved 73 ± 1.4 percent recovery for 100C DON spiked into 1kg corn grain. The limit of quantification for DON in ground corn grain was 50µg/kg for that particular study (32).
While GC ECD analysis of ergosterol is not commonplace, this technology has been used in the analysis of other environmental sterols (70). Previously described gas
62 chromatography methods were able to detect ergosterol and DON in single wheat kernels
(42, 112). To isolate ergosterol, a saponification step with potassium hydroxide (KOH) in methanol releases ergosterol from fungal membranes (42). Seitz et al. (1977) estimated that 38% of ergosterol is bound in the fungal cell membrane, while 62% is found freely in fungal cells. Dong et al (2006) achieved 93 – 95.6% extraction recovery for single wheat kernels spiked with ergosterol using GC mass spectroscopy. That study used 5% (w/v) KOH/methanol and incubated ground kernels at 80 °C for 6 hours for saponification. Water (0.5ml) and hexane (3ml) were added to extract ergosterol. The ergosterol extracts were desolvated and no clean up step was used. The method detection limit and method quantification limit were 18.5 and 55.6 ng/g (ppb), respectively (42).
Analyzing commodities for DON gluc is especially challenging and best practices or preferred methods have yet to be identified. DON gluc may potentially escape common DON detection methods as the addition of the glucose to the molecule may reduce specificity. Due to this challenge in DON gluc detection, conjugated toxins are also known as “masked mycotoxins” (18). Research has shown that enzyme linked immunosorbent assays specifically designed for DON will also detect DON gluc and acetylated derivatives of DON (191). However, these assays were unable to differentiate between these compounds. Hydrolyzing DON gluc and measuring the resulting DON content also provides means of determining total “DON potential” in a sample, but does not allow us to study DON and DON gluc separately.
Reported best extraction practices for DON gluc are conflicting. Trichothecene extraction from ground grain and quantification with liquid (HPLC) and gas chromatography (GC) has been extensively studied and evaluated (105). Extraction of
DON with acetonitrile water (84/16, v/v) provides the best extraction efficiency compared to other solvents (105). Berthiller et al. (2005) extracted DON with
63 acetonitrile water then extracted DON gluc from these extracts using Silica gel. Silica extracts were dried and DON gluc was resuspended in ethyl acetate/methanol (4/1, v/v).
Sansaya et al. (2008) reported that (50:50, v/v) methanol methylene chloride provided the better recovery of DON and DON gluc, while reducing matrix effects using LS UV MS.
This method also required use of a Strata X clean up column (148). Deoxynivalenol and
DON gluc were eluted from the column using of methanol and water. These studies employed mass spectroscopy to detect DON and DON gluc. Mass spectroscopy has been used to study DON gluc in harvested wheat grain (18, 19, 148). However, studies have reported matrix interference with DON gluc and lack of consistency when using liquid chromatography mass spectroscopy (88, 191). GC ECD analysis for DON gluc with acylation derivatization with HFBA has not been reported. However, HFBA derivatizes by conjugating fluorine molecules to hydroxyl groups, of which DON gluc has two. HFBA should provide adequate derivatization of DON gluc to permit GC ECD analysis.
The first objective of this research was to optimize protocols to extract DON,
DON gluc and ergosterol from a single piece of plant tissue: a wheat floret. A wheat floret includes all floral tissues and the vegetative tissue enveloping the developing kernel. Upon extraction, the second objective was to develop an analytical method to identify and quantify these compounds using GC ECD and derivatization by acylation with HFBA. The overall goal for the work described here was to combine extraction, derivatization and detection techniques to develop a GC ECD method which could be used to study F. graminearum infections in wheat heads. To the best of our knowledge, this methodology is the first designed to simultaneously detect ergosterol, DON and
DON gluc in single wheat florets.
64 Materials and Methods
Trichothecene Extraction
Single florets from FHB susceptible ‘Wheaton’ wheat heads inoculated with
Fusarium graminearum were weighed and placed in 2ml Eppendorf tubes with locking caps (Eppendorf North America; Westbury, NY). Two 5mm glass beads (Fisher
Scientific) were added to each tube and samples were ground by shaking at 30Hz for 2 minutes at room temperature with a tissue lyser (Tissue Lyser II, Qiagen Inc.; Valencia,
CA). The ground florets were transferred to individual 15 ml polypropylene tubes with
3ml 84/16 (v/v) acetonitrile/water (J. T. Baker, Mallinckrodt Baker, Inc.; Phillipsburg,
NJ) and were placed on an orbital shaker for 24 hours at room temperature and 220rpm.
All solvents used in this analysis were HPLC grade, unless otherwise noted. The extract was then decanted and the remaining floret tissue was stored at 4 oC. Acetonitrile water extracts were passed through clean up columns containing 0.5 g of 46.7/33.3/20 (m/m/m)
C 18 charcoal (EM Science; Gibbstown, NJ), aluminum oxide (J.T. Baker) and celite 545
(EM Science) between two polypropylene frits (For 15ml SPE reservoir, Grace Davison
Discovery Science; Deerfield,IL). Clean up columns were rinsed with 2ml of acetonitrile water. Cleaned extracts were dried under warm nitrogen gas and stored at
20 oC.
Ergosterol Extraction
Ergosterol was extracted from the same wheat floret tissue from which trichothecenes were previously extracted. Three milliliters of 1/1/5 (w/v/v) potassium hydroxide (Sigma Aldrich; St. Louis, MO), ethanol (Pharmco AAPER; Brookfield, CT) and methanol (J.T. Baker) were added to the floret tissue and incubated at 65 oC for 1 hour on a shaker (220 rpm). Samples were cooled to room temperature before 1ml of
65 water and 3ml of hexane (EMD Chemicals Inc; Gibbstown, NJ) were added to each tube.
Extracts were vortexed; the upper (hexane) layer was removed and placed into the same 8 ml vial containing the trichothecene extract from the same floret. Ergosterol extracts were desolvated under warm nitrogen gas. Finally the combined trichothecene and ergosterol extracts were stored at 20 oC.
Peak Identification and Standard Curves
Analytical standards of deoxynivalenol (Biopure; Tullin, Austria), deoxynivalenol 3 glucoside (Biopure) and ergosterol (Sigma Aldrich) were used to develop and calibrate this method. The following sets of dilutions were used to create standard curves: 0.10, 0.08, 0.05, 0.02 and 0.01 ng/ l DON; 2.0, 1.5, 1.0, 0.5 and 0.1 ng/ l DON gluc; 7.5, 5, 2.5, 1.0 and 0.5 ng/ l ergosterol. Retention time and peak identification of these compounds were confirmed by an outside lab using GC Mass
Spectroscopy (Chemical Ecology Laboratory; The Pennsylvania State University,
University Park, PA). An internal standard, 1 ng/ l (on column) mirex
(perchloropentacyclodecane, Sigma Aldrich), was added to each sample. Mirex has been used as an internal standard for GC methods designed to detect trichothecenes and ergosterol (42, 112). This internal standard served as a method performance check, as well as a calibration reference.
Known amounts of DON, DON gluc and ERG standards were also added to
“clean” floret extracts. These “clean” florets were from surface sterilized wheat heads grown in greenhouse conditions and were essentially disease and toxin free. By spiking a known amount of standard into a floret extract, standard peaks can be separated and identified from other compounds present in the floret matrix. The oven program was
66 optimized, by adjusting the oven temperature, to allow for peak separation and noise reduction.
Method Recovery and Hyphal Assessment
It is expected to experience some analyte loss during sample preparation steps.
For this reason a method recovery assessment was performed. Clean wheat florets were ground and spiked with DON, DON gluc and ERG standards which would result in
1.0ng/ul, 2.5ng/ul and 10ng/ul on column, respectively. Trichothecene and ergosterol extraction and cleaning protocols described above were followed. Estimated sample loss was calculated for each compound by comparing observed chromatographic signals to standards which were not subjected to the extraction, saponification and cleaning procedures.
To estimate average ergosterol content in fungal mycelium a combination of four
Fusarium graminearum isolates (R 07661, R 06979, R 07088 and R 09731; The
Fusarium Research Center, Penn State) was grown in potato dextrose broth (PDB). One hundred milliliters of PDB were added sterile 250 ml flasks before 5x10 5 macroconidia were inoculated. The liquid cultures were placed on a shaker (105rpm) and incubated at room temperature for 10 days. Mycelium was separated from the PDB using filter paper.
Hyphae were dried in a 50 oC oven for 24 hours. Approximately 125mg of dried mycelium was ground to a fine powder. Extraction of ergosterol was performed as described above.
Sample Derivatization
The acylation derivatization procedure using heptafluorobutyric anhydride
(HFBA, Campbell Science; Rockton, IL) was adapted from Croteau et al. (1994).
67 Through this process, a seven fluorine moiety is bound to a compound’s hydroxyl group(s), which renders the compound more volatile and improves detection by ECD.
Deoxynivalenol possesses 3 hydroxyl groups; DON gluc, 3 ADON and 15ADON have two hydroxyls each and ergosterol contains one hydroxyl group. To catalyze the derivatization reaction, 100ml of 2 mg/ml dimethyl aminopyridine (DMAP, Sigma
Aldrich) in toluene (EM Science) were added to each dried sample. Vials were briefly vortexed to re suspend the sample in DMAP. Fifty microliters of 2g/ml HFBA, the derivatizing agent, were added and the vials were incubated between 60 and 65 o for one hour to complete the reaction. Incomplete derivatization, resulting in multiple derivatives and multiple peaks, may result if the incubation time is shortened. The resulting derivatives were cooled to room temperature and 1.0ml of 3% sodium bicarbonate (J.T.
Baker) in water was added to neutralize the reaction. Vials were vortexed for 15 seconds, and 150 l of toluene was added to each vial to dilute the sample. The vials were vortexed another 15 seconds and the samples were transferred to amber colored auto sampler vials and capped with a Teflon septum (both, National Scientific; Rockwood,
TN). Samples were analyzed immediately following derivatization.
Instrumentation and Analytical Program Settings
Analyses were performed with a gas chromatograph with an electron capture detector (GC 17A, Shimadzu Scientific Instruments; Columbia, MD) and using
GCsolution software (Shimadzu Corporation, Tokyo). Ultra high purity helium was used as the carrier gas and zero grade nitrogen served as the make up gas. Stainless steel gas regulators were and copper tubing supplied gas flow to the chromatograph. A moisture trap (Hydro Purge II, Alltech; Lexington, KY) and oxygen trap (Oxy Trap,
Alltech) were inserted into the carrier and make up gas lines respectively, to ensure the
68 purity of the gas supplies. A split injection technique was used with AOC 20 auto sampler and injector (Shimadzu Scientific Instruments). The split ratio was 1:25. The injection port housed a deactivated glass insert containing glass wool and was maintained at 310 oC throughout analysis. A 15m capillary column (5% phenyl methyl silicone,
0.25mm inner diameter, 0.25µm film thickness) with a 5m guard column was used to develop this method (CA 5, Chromatography Associates; State College, PA and Rtx 5,
Restek; Bellefonte, PA). The oven temperature was initially held at 80 o for 1 minute, increased by 15 o/min to 140 o, slowly heated to 270 o by 2.5 o/min and held at the final temperature for 1 minute. The column flow was 1.3 ml/min and the ECD temperature was 310 oC.
Results
The GC ECD method describe above successfully and simultaneously detected trichothecenes and ergosterol. Amended protocols also successfully extracted naturally synthesized DON, DON gluc and ergosterol from wheat floret tissue. The extraction, cleaning and derivatization protocols are summarized in Figure 3 3. The oven program was able to separate the compounds of interest from a wheat floret matrix.
Deoxynivalenol, mirex, deoxynivalenol 3 glucoside and ergosterol eluted at 21.3, 37.6,
38.0 and 52.7 minutes, respectively (Figure 3 4). This method was also capable of detecting derivatives of DON, including 3 acetyl deoxynivalenol and 15 acetyl deoxynivalenol (Figure 3 4). Complete chromatographic analysis of a sample occurred in approximately 55 minutes.
Standard curves were developed using the commercially available standards
(Figure 3 5). These curves represented the relationship between analyte concentration and area under the corresponding chromatogram peak. This relationship was linear
69 between 0.01 and 0.10 ng/µl for DON; 0.05 and 7.50 ng/µl for DON gluc; and 0.100 and
10.00 ng/µl for ERG. Unknown concentrations of toxin or ergosterol in inoculated floret samples were then calculated using the equation of the standard curve line. These calculations and peak integrations were performed automatically by the GCSolution software at the end of each analysis. The limits of detection for DON, DON gluc and
ERG were 0.005, 0.025 and 0.050 ng/µl, respectively.
70
Figure 3 3. This method flow chart describes the steps taken to extract from floret tissue, clean and deri vatize deoxynivalenol, deoxyniv alenol 3 glucoside and ergosterol.
71
Figure 3 4. A) The GC ECD chromatogram shows peaks corresponding to deoxynivalenol ( DON, 21.1 min), the internal standard mirex (36.5 min), deoxynivalenol 3 glucoside ( DON gluc, 37.9 min) and 4) ergosterol ( ERG, 52.3 min), and B) acylated derivatives of deoxynivalenol, 15 acetyldeoxynivalenol (15 ADON, 26.8min) and 3 acetyldeoxynivalenol (3 ADON, 27.1min). Chromatogram B has been enlarged and cropped to show peak separation in detail.
72
Figure 3 5. The standard curves for deoxynivalenol, deoxynivalenol 3 glucoside and ergosterol depict the linear relationships between compound concentration and peak area.
73 A recovery analysis was performed to estimate the amount of compound retained in samples throughout the clean up and derivatization process. Standards were spiked into “clean”, non inoculated ‘Wheaton’ florets using the following concentrations:
1.0ng/µl DON, 2.5ng/µl DON gluc and 10.0ng/µl ERG. The percent recovery for DON,
DON gluc and ERG were 88 ± 8.8, 57 ± 4.1 and 37 ± 3.1, respectively (Table 3 1).
Fusarium graminearum was grown in culture to determine the average amount of ergosterol and trichothecenes in fungal mycelium. Five replicate analyses of 125 mg of fungal mycelium were performed. The chromatogram obtained from the fungal hyphae was similar to the chromatograms observed for the purified standards, in terms of peak shape and retention times (Figure 3 5). Using the standard curves, the mean ergosterol and deoxynivalenol concentrations and standard deviations were calculated. It was estimated that F. graminearum mycelium contained approximately 252.3 ± 14.1 ppm ergosterol and 0.095 ± 0.015 ppm DON when grown in PDB. Deoxynivalenol 3 glucoside was not detected, as cultures were grown in vitro, not in the presence of host tissue containing glycosyltransferase enzymes.
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. Percent Recovery Spiked Concentrations 1a 2 3 4 5 Mean SD b
1.0 ng/μl DON 80.3 78.6 93.8 99.5 89.3 88.3 8.87102
2.5 ng/μl DON-gluc 60.7 53.8 61.2 53.6 53.3 56.52 4.05179 10.0 ng/μl Ergosterol 36.7 33.8 35.9 36.6 42.1 37.02 3.070342 a Replications (in %) b Standard Deviation
74
Figure 3 6. The GC ECD chromatogram depicts the mean deoxynivalenol (DON, 21.2 minutes, 0.095ppm) and ergosterol (ERG, 52.7 minutes, 252.3 ppm) content of F. graminearum mycelium grown in potato dextrose broth. Mirex (37.9, 1.0 ng/ l) was used as an internal sta ndard.
Discussion
The sensitivity of t he described method is on par with previously reported methods for trichothecenes (32). Croteau et al. (1994) reported quantitation for DON as low as 4pg on column using a GC ECD method. Our method has a limit of quantit ation, the lower limit of the standard curve, of 5pg on column for DON. In a single wheat floret matrix, the limit of detection for DON is 50ng/ul. Mirocha et al. (1998) analyzed single kernels for DON using GC MS. They explained that it was too difficult to spike a mature kernel with toxin, but estimated recovery of DON from single kernels to be 90%
(112) . The florets used in the present study contained immature kernels and were relatively simple to spike with toxin and ergosterol. Rec overy using methods described here was 88% for 1.0ng/ l DON , which is comparable to recovery estimated by Mirocha at al. (1998) . High recovery was expected, as the extraction, clean up and GC ECD program were originally designed for type B trichothecene analysis (32) (105) .
75 Due to their “recent” discovery, few detection methods exist for masked mycotoxins, such as deoxynivalenol 3 glucoside. To date, very little work on DON gluc has taken place, especially on wheat from the United States. Sasanya et al. (2008) used liquid chromatography UV mass spectroscopy to study DON and DON gluc in U.S. spring wheat. A random sampling from grain found mean DON and DON gluc levels to be 1.4 ± 2.3 ppm and 0.2 ± 1.0 ppm, respectively (148). This LC methodology allowed for 96.4% and 70.0% recovery of DON and DON gluc, respectively (148). The sample preparation and GC method described here resulted in 37% recovery for 2.5ng/µl DON gluc. Small sample sizes, such as single florets, inevitably have lower percent recoveries, than larger sample sizes, such as several grams of grain. The two step extraction of trichothecenes and ergosterol and derivatization protocols also allow for some sample loss. Another possibility for low DON gluc recovery was analyte loss during the cleaning process. An alternative clean up column that has reduced affinity for DON gluc could be used in the future, such as the Strata X column used by Sasanya et al. (2008).
Analysis of DON gluc in single kernels or florets has not been reported until now.
To our knowledge, there are no published reports of GC ECD methods for detecting ergosterol. However, acylation derivatization and GC ECD analysis have been used to study other sterols (70). Dong et al. (2006) studied ergosterol in mature barley kernels using GC MS. Barley kernels were spiked with different levels of ergosterol standard and found percent recovery to be between 88% and 97% (42). In general, greater concentrations of standard result in lower percent recoveries. In the present study, the percent recovery for 10ng/ul ergosterol was 37%, when spiked into an immature wheat floret. The double extraction protocols, for trichothecenes and ergosterol, and saponification or sterols may lead to significant ergosterol loss. GC ECD with HFBA derivatization may not be ideal for ergosterol analysis as ergosterol only has
76 one hydroxyl site for derivatization. This may also decrease ergosterol quantification sensitivity. However, these methods do allow for qualitative analysis and detection of fungal biomass in single wheat florets.
By simultaneously detecting all three compounds, we have created a means of monitoring fungal colonization, toxin production/translocation and toxin conjugation in host tissue. This is a powerful tool that will allow for qualitative and semi quantitative characterization of Fusarium infections in wheat heads. While other detection methods for trichothecenes and ergosterol exist, very few can detect both compounds from a single piece of plant tissue. Enzyme linked immunosorbent assays (ELISA) are very sensitive but these tests may not be able to differentiate between DON, acylated derivatives or
DON 3 glucoside, as they have similar structures. Furthermore, separate ELISA tests and multiple pieces of plant tissue would have to be used to analyze DON and ergosterol.
Molecular methods are designed to quantify expression of fungal trichothecene and ergosterol synthesis genes. However, molecular tools would not be able to detect the translocation of DON alone within the wheat head. While a PCR based method would be able to detect trichothecene gene expression in the pathogen and glucosyl transferase expression in the host plant, it would not be possible to quantify DON 3 glucoside formation in the host. Mass spectroscopy, as in GC MS, has been used to detect DON,
DON gluc and ERG in a wheat kernels or flour. An advantage of using GC MS is its specificity, as it identifies compounds based on their specific molecular mass. The derivatization step is also not required for GC MS analysis. By using GC ECD, the user can only identify compounds by comparing their retention time to that of known standards. However, in the absence of mass spectroscopy, the GC ECD method and
HFBA derivatization protocols described here provide efficient and sensitive means of identifying DON, DON gluc and ERG in single wheat florets.
77 This analytical method was designed to specifically indentify DON, DON gluc and ERG in single wheat florets. In the future this method may be adapted for analysis of these compounds within other wheat substrates, such as single kernels, entire heads or flour. Previous studies have examined ergosterol and trichothecene content in single kernels (42, 112). However, research has shown that greater levels of trichothecenes accumulate in the bran and floral tissues (162). By analyzing entire florets, a more complete snapshot of infection development and toxin translocation is captured. This research tool may be used to study the pre and post harvest concentration of toxins and fungal growth in many commodities. In fact, this method has also proven successful for analyzing ergosterol content of F. graminearum mycelium. In the future, protocols will be adapted to analyze fungal biomass in corn seedlings.
78
Chapter 4
THE EFFECTS OF TEMPERATURE A D HOST O FU GAL
BIOMASS, DEOXY IVALE OL ACCUMULATIO A D
CO JUGATIO I SI GLE WHEAT FLORETS
Introduction
Fusarium Head Blight (FHB) is a devastating disease of wheat in terms of yield and grain quality (106). This disease is caused by a complex of Fusarium species, predominantly F. graminearum in the United States. The fungus infects the developing kernels and causes pre mature senescence of the spike tissue. Kernels from affected wheat may be shriveled or covered with white or pink mycelium (16). These fungi also produce mycotoxins, such as the trichothecene deoxynivalenol (DON), which are toxic to humans and animals when ingested in small doses (129, 131). The mode of toxicity lies in the ability of DON to bind to eukaryotic ribosomes, inhibiting protein synthesis (110).
In general, FHB symptoms observed in the field or on harvested kernels are positively correlated with DON levels in the grain (95, 123). However, significant levels of DON
(greater than the FDA guideline of 2ppm in raw grain) are not always synonymous with visible symptoms (162). Many theories have been developed to explain this phenomenon, including effects of environment, infection timing, DON translocation and/or conjugation (18, 30, 33) (Chapter 2). Yet, evidence to support these theories is lacking, as well as the tools to simultaneously study mycotoxin accumulation and fungal growth during infection.
Fusarium graminearum infections can take place from anthesis through the hard dough stages of wheat development (35, 62) (Chapter 2). Ascospores and/or
79 macroconidia inoculate wheat heads and begin to germinate shortly thereafter. Pritsch et al. (2000) showed macroconidia germinating on the glume surface within 6 hours after inoculation. Generally, F. graminearum does not directly penetrate the external floret tissue (139). Instead, mycelium colonizes the surface of the glume until it reaches an opening, such as a stomate (139). The pathogen may also colonize the interior of the glume tissue. Once inside the wheat floret, hyphae can easily colonize and directly penetrate the host through the stigma and ovary (76). Subcuticular hyphal growth has been reported to occur between 2 and 3 days post inoculation (69, 139). Hyphae often penetrate the cell walls through pits or plasmadesmata (69). Then the hyphae travel inter and intra cellularly towards the rachis node (76). Kang and Buchenauer (2000) found a difference in the time it took F. culmorum to reach the rachis between resistant and susceptible wheat cultivars. In susceptible lines hyphae reached the rachis and vascular tissue within 4 to 5 days after inoculation. In a moderately resistant cultivar, it took 8 to
10 days. Once the hypha reaches the rachis and vascular tissue, greater pathogen growth is observed below the inoculated floret (7).
Mycotoxin production occurs soon after spore germination and is excreted by fungal hyphae to precede growth in planta (75). Deoxynivalenol has been detected on lemma and ovary tissue with 36 hours after inoculation with F. culmorum (75). In wheat cells, DON was found on cell walls and chloroplasts and were frequently associated with ribosomes and the endoplasmic reticulum (76). Kang and Buchenuaer (1999) found
DON in uninfected vascular bundles. Ten days following inoculation of a central spikelet, no fungal hyphae were found in the third spikelet or vascular tissue above the inoculation point (75). However, DON was found in the vascular tissue at this position and to a lesser extent on the caryopsis and within the endosperm of the developing kernel.
This strongly suggests that DON is able to translocate through the xylem and phloem to
80 regions of the wheat head uncolonized by the pathogen. Mycotoxin accumulation patterns were also studied by Savard et al. (2000). However, this study found more DON accumulation in florets below the point of inoculation than above (149). Deoxynivalenol translocation was suggested to occur as early as four days post inoculation (149). DON accumulates in greater concentrations in the rachis and peduncle (1000 to 1200ppm) than in spikelets and florets (500 to 600ppm) (149). Argyris et al. (2005) observed higher levels of F. graminearum infection below the point of inoculation (7). Differences in wheat head infection patterns and DON accumulation trends may differ between wheat cultivars. However, few studies have simultaneously examined DON translocation and fungal colonization of wheat heads during grain development.
In addition to host genotype, FHB development and mycotoxin accumulation is influenced by environment. As discussed in Chapter 2, moisture and rain events are important for distributing inoculum and promoting infections. Temperature is also critical to F. graminearum biology and toxin production. Sutton (1985) suggested FHB epidemics have an upper limit of 32 oC and a lower limit of 15 oC. In vitro studies show the ideal temperature for F. graminearum growth is 28 oC (14), while optimal DON production occurs around 22 oC (104). However, toxigenic fungi may respond to abiotic stress, such as lower temperatures (15 oC), by increasing mycotoxin production (155).
Yet, lower temperatures may inhibit colonization and infection of host tissues. A study of Fusarium verticillioides (formerly known as F. moniliforme ), a fumonisin producer and corn pathogen, found that ergosterol concentration was affected by temperature (4).
Alberts et al. (1990) grew the fungus in corn cultures and analyzed ergosterol concentrations every two weeks. The initial growth rates after 2 weeks of incubation were greater at 30 o than 25 or 20 oC. Furthermore, incubation at 25 oC produced more ergosterol (fungal biomass) than cultures incubated at 20 oC during the first two weeks.
81 However, after 4 to 6 weeks of incubation, ergosterol concentrations in the 25 and 20 oC cultures reached a similar maximum level (approximately 5g/kg). This suggests that temperature plays a significant role in ergosterol concentration during the earliest stages of fungal growth, but less so as the fungus matures. This may also be true for the earliest stages of FHB disease development.
In addition to deoxynivalenol, the presence of conjugated mycotoxins in wheat has garnered recent attention (18, 148). Conjugated DON, such as deoxynivalenol 3 glucoside (DON gluc), is produced in planta by glucosyltransferase enzymes (137).
Lemmens et al. (2005) discovered that the ability to form conjugated DON is linked to
FHB1, the major QTL for moderate FHB resistance. It remains unclear whether FHB1 encodes or regulates the glycosyltransferase enzyme (96). Recent research indicates that the presence of DON gluc in wheat and barley is common, however the ratio of DON to
DON gluc is variable (18, 88, 148, 191). The relationship between DON and DON gluc requires further study. It is unclear which biotic and abiotic factors play a role in DON gluc formation in wheat heads. Furthermore, very little is known about DON gluc accumulation during FHB infections.
The goal of this research was to employ the analysis method described in Chapter
3 as a tool to study fungal growth (ergosterol), DON translocation and DON gluc formation during the early stages of FHB disease development. By analyzing single florets, colonization and toxin accumulation patterns within individual wheat heads were characterized. The effects of host resistance and temperature (15 versus 22 oC) on F. graminearum colonization of wheat heads and mycotoxin translocation were also examined. It was hypothesized that the cooler temperature (15 oC) would limit fungal growth following point inoculations of wheat heads but allow for DON production and translocation, leading to uncolonized florets containing DON. Twenty two degrees was
82 hypothesized to stimulate colonization throughout the spikes as well as DON accumulation. A moderately resistant cultivar was expected to accumulate less DON and ergosterol than an FHB susceptible cultivar.
Materials and Methods
Inoculum Preparation
Four Fusarium graminearum isolates isolated from Pennsylvania (R 07661, R
06979, R 07088 and R 09731; Fusarium Research Center, University Park, PA) were selected for this study. Mung bean agar (Dr. Ruth Dill Macky, personal communication) was prepared by boiling 40g of mung beans (‘OK2000’, Oklahoma Crop Improvement
Association) in one liter of distilled water. The beans were removed from the heat as soon as they began to split open (approximately 20 minutes). The bean broth was filtered through a cheesecloth lined B üchner funnel into a flask. The beans were discarded and
15g of agar were added to the bean broth. The total volume was brought up to one liter with distilled water and was sterilized. Isolates, first cultured on carnation leaf agar, were inoculated onto the agar plates. Plates were incubated for seven to ten days at 25 oC, under a twelve hour diurnal cycle using fluorescent and black lights. Macroconidia from these isolates were harvested by scraping a sterile microscope slide across the agar surface and filtering the inoculum through sterile cheesecloth. Macroconidia were quantified using a hemacytometer and diluted with sterile water to 10 4 spores/ml.
Inoculum was stored at 20 oC until use.
Wheat Production in the Greenhouse
Two hard red spring wheat cultivars were used in this study. ‘Alsen’ is moderately resistant to FHB, while ‘Wheaton’ is a susceptible cultivar. ‘Alsen’ is
83 currently a popular, high yielding wheat variety grown in the upper mid west regions of the U.S. (52). Wheat seeds from each cultivar were surface sterilized with 2% bleach.
Sterile filter paper was placed in the bottom of 250ml glass beakers. The filter paper was moistened with sterile water and wheat seed from each cultivar was scattered on top of the filter paper. The beakers were loosely enclosed in clear plastic bags. The wheat seeds were allowed to imbibe water and germinate for five days or until the coleoptiles reached approximately 5cm in height. The seedlings were then potted with Metro Mix
360 (Sun Gro Horticulture Canada Ltd; Seba Beach, Alberta) in the plant pathology greenhouses (The Pennsylvania State University, University Park, PA). Greenhouse conditions varied from 18 to 35 oC, and potting mix was kept moist with frequent watering, as needed. After the seedlings were established and had reached the 3 to 4 leaf stage (Zadoks 14), a soil drench with 2 grams per liter imidacloprid (Admire 2E, Bayer
Crop Science; Research Triangle Park, NC) was performed to prevent aphid infestations.
Around the same time, a weekly fertilization regimen with 20% N, 20% P2O5, and 20%
K2O (Peter’s Professional All Purpose Plant Food, Spectrum Brands; Alpharetta, GA) began and continued until the plants reached the boot stage (Zadoks 45). Bamboo stakes were used to stabilize the growing plants.
Point Inoculations
Upon mid anthesis (Zadoks 65), wheat plants were moved from the greenhouse to temperature controlled Conviron growth chambers (Controlled Environments Limited;
Winnipeg, Manitoba, Canada). A wheat spikelet (two florets) (Figure 4 1) in the middle of the wheat head was selected for inoculation with F. graminearum . Each floret was inoculated with one 10 l droplet of 10 4 macroconidia/ml. This was performed by gently separating the lemma from the palea and placing a pipette tip inside each floret. The
84 droplet of inoculum was expelled in side the floret cavity containing the female floral components (Figure 4 2). A small strip of moistened, sterile paper towel was wrapped around the base of the wheat head. The inoculated wheat heads were bagged for 48 hours to increase humidity and to facilitate colonization and infections (Figure 4 2).
Approximately half of the inoculated ‘Wheaton’ and ‘Alsen’ plants were placed into a growth chamber set at 22 oC. The other half were placed in to a 15 oC chamber.
Both chambers were programmed to complete a 12 hour diurnal cycle and maintain 80% relative humidity. Three replications of this experiment were performed between 2008 and 2009.
Figure 4 1. Wheat floret anatomy is depicted in this photograph of a spikelet . Between the lemma and palea, the feathery stigma and yellow anthers of the flower can be observed. (Photograph credit: http://www.castonline.ilstu.edu/ksmick/150/150mflower/150whspik.JPG)
85
______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 mid anthesis. 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 spikele t (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 p lated on Nash agar and the other was used for chromatographic analysis.
86 Floret Harvests
Entire wheat heads were harvested 2, 4, 6, 8, 10 and 12 days post inoculation.
The rachis was cut just below the base of the wheat head and heads were placed in 250ml flasks. Floret symptoms, such as lesions, discoloration or bleaching, were recorded.
Heads were surface sterilized with 2% bleach, with a few drops of Tween20
(polyoxyethylene sorbitan monolaurate) to serve as a surfactant. Heads were then rinsed twice with sterile water. Immediately thereafter, spikelets above and below the point of inoculation were harvested. Due to variable head size, harvested florets were limited to four above (+1 to +4) and below ( 1 to 4) the point of inoculation (floret 0) (Figure 4 3).
One floret from each harvested spikelet was placed on Nash agar (98, 115), a Fusarium selective media, to provide incidence data. Nash plates were sealed with Parafilm and incubated at 25 oC, under a 12 hour diurnal cycle. The presence of Fusarium , growing from harvested florets, was detected and recorded (0 = no fungus, 1 = Fusarium present/floret infected) approximately 5 10 days after plating.
The second floret from the harvested spikelets was reserved for fungal biomass and mycotoxin analysis. These florets were weighed and placed in individual sample tubes (Eppendorf). Two glass beads were added to each tube and the florets were homogenized (30Htz, 2 minutes) using a Tissue Lyser II (Qiagen Inc.; Valencia, CA).
The ground floret tissue was centrifuged at 13,000 rpm for two minutes and stored at
20 oC until trichothecene extraction.
Sample Preparation and Analysis
To assess fungal growth and trichothecene translocation, a gas chromatography method was developed (Chapter 3). Fungal growth was quantified using the biomarker ergosterol, a sterol unique to fungal cell membranes. Gas chromatography with electron
87 capture detection (GC 17A, Shimadzu Scientific Instruments; Columbia, MD) was used because of its sensitivity and rapid analysis capabilities. A fifteen meter 5% phenyl methyl silicone column (0.25mm inner diameter) along with a five meter guard column was used in this study (CA 5, Chromatography Associates, State College, PA; and Rtx5,
Restek, Bellefonte, PA). Ergosterol (Sigma Aldrich; St. Louis, MO), deoxynivalenol and deoxynivalenol 3 glucoside (Romer Labs, Union, MO) standards were used to initially develop and calibrate the method.
Three milliliters of acetonitrile water (84/16) were added to the ground floret tissue to extract trichothecenes. Samples were placed on a shaker for approximately 24 hours at room temperature. The resulting extract was filtered through a charcoal alumina column. The clean extract was dried under warm nitrogen gas and stored in the freezer.
To extract ergosterol from the same floret tissue, 3 milliliters of 1:1:5 (m/v/v) potassium hydroxide, ethanol and methanol were added to each tube. Floret samples were placed on a shaker at 65 oC for one hour. This process releases ergosterol esters bound to fungal cellular membranes. One milliliter of water and 3ml of hexane were added to each tube and vortexed briefly. The upper layer, containing any available ergosterol, was removed and placed into vials containing the dried trichothecene extracts. Vials were dried under warm nitrogen gas for a second time and stored at 20 oC until analysis.
Samples were derivatized with 50 l of hepta fluorobutyric anhydride as described in Croteau et al. (1994). One hundred microliters of 2 mg/ml dimethylaminopyridine were added to each dried sample (32). The column oven program was based on a previously developed GC ECD method for DON, with some modifications (112).
Briefly, the column was held at 80 oC for 1 minute, increased to 270 o by 2.5 o/min and held at 270 o for 1 minute. An internal standard, mirex, was added to each sample to serve as a check and reference peak. A standard sample, including known concentrations of DON,
88 DON gluc and ERG, was included with each batch to ensure the accuracy of compound retention times and standard curves.
Statistical Analysis
PROC GLM of SAS (Version 9.2, SAS Institute; Cary, NC) was used to characterize the effects of cultivar, temperature and harvest date on F. graminearum incidence. Incidence, a binary measurement, was recorded for each harvested floret (“1” for the presence of Fusarium , “0” for no Fusarium growth on Nash agar). Average
Fusarium incidence per wheat head was calculated. For example, if four out of the nine harvested florets were positive for Fusarium , then the percent incidence for this wheat head would be 44.4%. Three total replications were used in incidence analysis. PROC
GLM was also used to examine the effect of cultivar, temperature, harvest date, floret position and their interactions on fungal biomass and mycotoxin accumulation in infected wheat heads. Two complete replications were used for DON and ERG analysis. The
Tukey multiple comparison procedure was performed to estimate pair wise comparisons of least square means using the family confidence coefficient of 95%.
Results
Preliminary experimental work demonstrated that greenhouse grown wheat reached anthesis about 8 12 weeks after planting. The variability was due to uncontrollable greenhouse temperatures and the duration of ambient sunlight. Warmer temperatures and long days promoted wheat growth and maturation. Nevertheless,
‘Wheaton’ and ‘Alsen’ reached anthesis at approximately the same time. Preliminary work also determined that 80% relative humidity in the growth chambers facilitated infections. One hundred percent relative humidity caused fungal hyphae to proliferate on
89 the outside of the glumes. Although this is occasionally observed, aerial mycelium is not typical of FHB infections in the field. The inoculation and incubation methods described facilitated infections and induced symptoms on wheat heads. Earliest symptoms included slight browning of glume tissue and small water soaked lesions. These symptoms were observed as early as 2 days post inoculation (dpi) in the susceptible ‘Wheaton’, incubated at 15 and 22 oC. Minor necrosis first appeared in ‘Alsen’, the moderately resistant cultivar, by 4dpi under both temperature treatments. Typical FHB bleaching symptoms occurred in both cultivars by 8dpi. Necrosis at the floret node and brown developing kernels were also observed. Symptoms beyond the inoculated floret were observed by
10dpi in both cultivars.
Incidence of Fusarium in Wheat Florets
Preliminary incidence data, from Nash agar plates, demonstrated that F. graminearum was able to colonize the +4 and 4 florets of both ‘Alsen’ (moderately resistant) and ‘Wheaton’ (susceptible) by 12 days post inoculation. This result was observed at both 15 and 22 oC temperature treatments. Since the primary goal of this work was to study the translocation of DON and fungal growth during the early infection stages, data collection was limited to 10 days post inoculation. To assess the effects of cultivar, temperature and harvest date (dpi) on Fusarium incidence a general linear model was employed (R 2 = 0.604, P = 0.0032).
The effects of incubation temperature (P = 0.0005) and harvest day (incubation time) (P = 0.0001) on Fusarium incidence were significant (Table 4 1). Florets incubated at 22 oC experienced 51.9% Fusarium incidence across both cultivars, compared to the 15 oC treatment which had 30.0% incidence. In general, incidence of
90 Fusarium in florets increased with the length of incubation time. However there were no significant increases in incidence between 6, 8 and 10dpi (Table 4 2).
While ‘Wheaton’ experienced greater floret colonization per wheat head (43.7%) compared to ‘Alsen’ (38.1%), the effect of cultivar was not significant (Table 4 1). This suggests that host resistance possessed by ‘Alsen’ was not a factor in inhibiting growth of
Fusarium following inoculation. Susceptibility to FHB possessed by ‘Wheaton’ was also not a factor for promoting fungal growth, during the early stages of infection.
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 oC and harvest at 2, 4, 6, 8,and 10 days post inoculation.
Source df a F Value b Pr > F c Cultivar 1 0.91 0.3446 Temperature 1 14.15 0.0005 Day 4 7.73 0.0001 Cultivar x Temperature 1 0.1 0.7515 a Degrees of freedom b F statistic associated with the analysis of variance c Probability associated with the F test.
Table 4 2. Fusarium incidence for each harvested day post inoculation, calculated across all cultivars, temperature treatments and individual florets.
Days Post-Inoculation Incidence (%) * 2 15.7 c** 4 27.8 bc 6 51.9 ab 8 53.7 ab 10 55.6 a *Incidence of Fusarium on Nash agar, as calculated per wheat head ** Different letters indicate significant differences (P ≤0.05) between lsmeans as determined by the Tukey procedure.
91 Deoxynivalenol Translocation
A general linear model (R 2 = 0.549, P = 0.0859) was employed to examine the effects of cultivar, temperature, day and cultivar temperature on DON accumulation. The factors of harvest day (P = 0.0364) and the cultivar temperature interaction (P = 0.0340) were significant to the model (Table 4 3), suggesting these factors influence DON production and translocation in wheat heads.
Although not significantly different, the overall least square mean for DON accumulation at 15 oC (0.125ng/µl) was greater than that of wheat incubated at 22 oC
(0.085 ng/µl). There was also no significant difference between overall DON levels in
‘Alsen’ (0.104ng/µl) and ‘Wheaton’ (0.106ng/µl) across both temperatures and all harvest days. However, the interaction between temperature and cultivar was significant to the model (Table 4 3). The greatest amount of DON was detected in ‘Alsen’ wheat heads incubated at 15 oC (Table 4 2). While the least amount of DON was detected in
‘Alsen’ incubated at 22 oC.
Harvest day was also found to be significant to the model, and in general, DON accumulation in wheat heads increased over time (Table 4 5). However, significant differences in DON levels between harvest days levels were not detected using the
Tukey Kramer test. In general, DON accumulation increased with incubation time.
There was a small decline or lack of increase between 8 and 10dpi.
While the cultivar temperature day floret interaction was not incorporated into to the DON GLM model, it does provide a timeline of DON production and translocation during the early stages of FHB infections. Deoxynivalenol production was observed in both wheat cultivars and in both temperature treatments by 2dpi (Figure 4 5).
Translocation of DON to florets above and below the inoculation point was also observed
92 by 2dpi. In ‘Alsen’, heads were fully contaminated with DON by 4dpi when incubated at 15 oC. ‘Wheaton’ heads were not fully contaminated until 6dpi (22 oC incubation).
Table 4 3. The effects of fixed factors on DON accumulation in ‘Alsen’ and ‘Wheaton’ wheat florets incubated at 15 or 22 oC and harvested by 2, 4, 6, 8 or 10 days post inoculation. Source df a F Value b Pr > F c Cultivar 1 0 0.9687 Temperature 1 0.81 0.3706 Day 4 2.62 0.0363 Cultivar x Temperature 1 4.57 0.034 a Degrees of freedom b F statistic associated with the analysis of variance c Probability associated with the F test.
Table 4 4. Mean deoxynivalenol accumulation calculated for each cultivar*temperature interaction, across all harvest days and florets. Cultivar Temperature a Deoxynivalenol b Alsen 15 0.172 a* 22 0.036 a
Wheaton 15 0.078 a 22 0.134 a a Incubation temperature post inoculation in oC. b Deoxynivalenol least square means in ng/µl. *Different letters indicate significant differences (P ≤0.05) between lsmeans as determined by the Tukey Kramer test.
Table 4 5. Mean deoxynivalenol accumulation for each harvested day post inoculation, calculated across all cultivars, temperature treatments and individual florets.
Days Post-Inoculation Deoxynivalenol * 2 0.020 a** 4 0.035 a 6 0.093 a 8 0.193 a 10 0.185 a * Deoxynivalenol least square means in ng/µl. **Different letters indicate significant differences (P ≤0.05) between lsmeans as determined by the Tukey Kramer test.
93
Figure 4 4. Mean deoxynivalenol concentrations in single florets following point inoculatio n of floret 0 of moderately resistant ‘Alsen’ (A) and susceptible ‘Wheaton’ (B). Florets were incubated at 15 or 22 oC, harvested 2, 4, 6, 8 and 10 days post inoculation and estimated by gas chromatography electron capture detection.
94 Fungal Biomass Estimated by Ergosterol
A general linear model was also used to examine the effects of cultivar, temperature, day and cultivar temperature ergosterol accumulation in wheat heads (R 2=
0.649, P <.0001). Temperature, day and cultivar temperature were significant ( P ≤ 0.05) in the model (Table 4 6). Overall ergosterol levels were slightly higher in ‘Wheaton’
(0.710ng/µl), the more susceptible cultivar, than in ‘Alsen’ (0.604ng/µl). However these means were not statistically different. The effect of temperature was significant (P =
0.0002), as heads incubated at 22 oC contained an overall average of 0.857ng/µl ergosterol compared to 15 oC (0.457ng/µl ergosterol). Overall, fungal biomass increased over time, as florets harvested earlier contained less ergosterol than florets harvested after prolonged incubation (Table 4 7). The greatest concentration of ergosterol was found in florets harvested 8dpi. There was a significant ( P = 0.0021) decrease in fungal biomass observed between days 8 and 10 post inoculation.
The interaction between cultivar and temperature was also significant in the model. Greater levels of ergosterol were observed in both cultivars at 22 oC than at 15 oC
(Table 4 8). This observation was significant ( P <.0001) for ‘Alsen’ only, as wheat incubated at 22 oC contained over three times the ergosterol as wheat incubated at 15 oC.
Significant interactions were also found between temperature and day of harvest (Table
4 6). While these interactive effects were significant to the ergosterol model, few significant differences were found between each interaction, likely due to the small number of replications in this analysis.
Again, the four way interaction between cultivar, temperature, harvest day, and floret position provides a means to follow fungal colonization of wheat heads (Figure 4
5). In ‘Wheaton’, the more susceptible cultivar, wheat heads incubated at 15 oC were
95 fully colonized by 6dpi. In ‘Alsen’, the first instance of a fully colonized head was observed by 8dpi (22 oC).
Table 4 6. The effects of fixed factors on fungal growth and colonization of ‘Alsen’ and ‘Wheaton’ wheat florets incubated at 15 or 22 oC and harvested by 2, 4,6, 8 or 10 days post inoculation, as estimated by ergosterol. Source df a F Value b Pr > F c Cultivar 1 1.02 0.3129 Temperature 1 14.66 0.0002 Day 4 23.21 <.0001 Cultivar x Temperature 1 13.07 0.0004 a Degrees of freedom b F statistic associated with the analysis of variance c Probability associated with the F test.
Table 4 7. Mean ergosterol accumulation for each harvest day, calculated across all cultivars, temperature treatments and individual florets. Days Post-Inoculation Ergosterol * 2 0.020 c** 4 0.035 c 6 0.093 bc 8 0.193 a 10 0.185 b *Ergosterol least square means in ng/µl. ** Different letters indicate significant differences (P ≤0.05) between lsmeans as determined by the Tukey procedure.
Table 4 8. Mean ergosterol accumulation estimated for each cultivar*temperature interaction, across all harvest days and florets. Cultivar Temperature a Ergosterol b Alsen 15 0.215 b* 22 0.994 a 0.699 Wheaton 15 0.721 a 22 0.134 a a Incubation temperature post inoculation in oC. b Ergosterol least square means in ng/µl. *Different letters indicate significant differences (P ≤0.05) between lsmeans as determined by the Tukey procedure.
96
Figure 4 5. Mean ergosterol concentrations i n single florets following point inoculation of floret 0 of moderately resistant ‘Alsen’ (A) and susceptible ‘Wheaton ’ (B). Florets were incubated at 15 or 22 oC, harvested 2, 4, 6, 8 and 10 days post inoculation and estimated by gas chromatography electron capture detection.
97 Deoxynivalenol-3-glucoside Synthesis in Wheat Heads
To date, only qualitative data from one replication of this experiment has been collected regarding the presence of DON gluc in wheat heads. Both ‘Alsen’ and
‘Wheaton’ produced this conjugated mycotoxin by 6 days following inoculation (Figure
4 6). Deoxynivalenol 3 glucoside was first detected in the inoculated floret, which is where the most DON was usually found. On average, ‘Wheaton’ produced more DON gluc (0.021ng/µl) than ‘Alsen’ (0.011ng/µl). ‘Wheaton’ also produced DON gluc with higher frequency than ‘Alsen’. Out of the florets analyzed 18.9% of ‘Wheaton’ florets contained DON gluc, while 10% of ‘Alsen’ florets contained DON gluc. By 10dpi, the majority of ‘Wheaton’ florets analyzed contained some DON gluc. The ratio of DON gluc to DON ranged from 0 to 332.7% (Figure 4 9). In three cases, the concentration of
DON gluc per wheat head exceeded the concentration of DON. To our knowledge this is the first report of DON gluc detection in single wheat florets.
98
Figure 4 6. Deoxynivalenol 3 glucoside concentration in single florets following point inoculatio n of floret 0 of moderately resistant ‘Alsen’ (A) and susceptible ‘Wheaton’ (B). Florets were incubated at 15 or 22 oC, harvested 2, 4, 6, 8 and 10 days post inoculation and estimated by gas chromatography electron capture detection.
99 Table 4 9. Mean deoxynivalenol 3 glucoside and deoxynivalenol accumulation and the ratio between the two compounds in wheat heads. Day Post- Ratio DON- Cultivar Inoculation Temperature a DON-gluc b DON c gluc : DON Alsen 2 15 0.0000 0.0086 0.000 22 0.0000 0.0059 0.000 4 15 0.0000 0.0442 0.000 22 0.0000 0.0187 0.000 6 15 0.0187 0.0521 0.359 22 0.0221 0.0281 0.786 8 15 0.0000 0.2055 0.000 22 0.0610 0.0203 3.001 10 15 0.0000 0.2376 0.000 22 0.0341 0.0103 3.327
Wheaton 2 15 0.0000 0.0057 0.000 22 0.0000 0.0076 0.000 4 15 0.0000 0.0656 0.000 22 0.0000 0.0112 0.000 6 15 0.0000 0.0299 0.000 22 0.0450 0.3021 0.149 8 15 0.0128 0.1302 0.098 22 0.0000 0.0642 0.000 10 15 0.0804 0.0393 2.044 22 0.1119 0.1177 0.950 aIncubation temperature following point inoculations of a central spikelet with F.graminearum bMean deoxynivalenol 3 glucoside concentration per wheat head (ng/µl) cMean deoxynivalenol concentration per wheat head(ng/µl)
Discussion
This work demonstrated the successful extraction and simultaneous analysis of deoxynivalenol and ergosterol from wheat florets. Using these methods, it was possible to study early stages of F. graminearum colonization of wheat heads. Semi quantitative data were collected regarding fungal colonization and mycotoxin accumulation during early Fusarium Head Blight disease development. Fungal response to host genotype and temperature was studied over a ten day period. Wheat heads were inoculated using a point injection technique, which involved gently inserting a droplet of F. graminearum
100 macroconidia between the lemma and palea of both florets of a central spikelet. Previous studies have studied deoxynivalenol translocation or assessed Type II resistance
(resistance to pathogen spread) following a point inoculation (7, 76, 149). However, few studies have simultaneously studied fungal growth and toxin translocation in wheat heads.
Previous work has attempted to characterize the relationship between Fusarium and trichothecenes in wheat. In harvested wheat grain, ergosterol has been used as a marker to predict DON concentrations (91). Miller and Young (1985) found a significant correlation between ergosterol and DON during wheat development. It was determined that both DON production and fungal growth increase until 6 weeks post inoculation.
Between 6 and 9 weeks post inoculation DON levels decreased and ergosterol levels remained unchanged (111). This is likely due to decreased fungal growth and detoxification or conjugation of DON. Indeed, the relationship between fungal growth and DON accumulation during kernel development is constantly changing due to host biology and the environment, as seen in the present study. Higher concentrations of
DON and ergosterol are typically found in the chaff and rachis than in the kernels (162).
Snijders and Krechting (1992) found DON translocation to occur between the chaff and the developing kernel. However, ergosterol spread into the kernel was inhibited, leading to grain with higher levels of DON, without fungal biomass (164). Kang and Buchenauer
(1999) also detected the presence of DON in the vascular tissue of wheat heads, in the absence of the pathogen. This suggests that DON is translocated to parts of the wheat heads uncolonized by Fusarium. The present study confirms DON translocation, as the toxin was found in florets, in the absence of ergosterol, the fungal biomarker.
Furthermore, incidence assays confirmed Fusarium was not present in these DON contaminated florets, especially during the early stages, such as 2 days post inoculation.
101 Argyris et al. (2005) examined fungal growth patterns following a point inoculation of a central spikelet and found F. graminearum to colonize portions of more resistant wheat heads below the inoculation point with greater frequency than florets above. In the most susceptible cultivar, the pathogen colonized the wheat head above and below the point of inoculation. This pattern was not observed in the present study.
Highest levels of ergosterol were usually found in the inoculated floret; however ergosterol was detected in regions above and below the inoculated floret. Moreover, in the earliest harvest periods (2 to 4dpi), ergosterol was found more frequently and in greater concentrations in the upper florets, than those below the inoculated floret. The differences in these findings may be due to host genotype or because of differences in study protocols. Argyris et al. (2005) dissected wheat heads at kernel maturity and individually analyzed floral components, the seed and the rachis for the presence of F. graminearum . Their work provides evidence that the pathogen enters the rachis and uses vascular tissue to colonize wheat heads. The present study examined the early stages of infection. Due to the design of this study, it cannot be confirmed how F. graminearum colonized the wheat heads used in this study. The pathogen may have colonized florets by growing over the external surfaces of the wheat head. Research has shown that FHB pathogens are able to reach the rachis by 5dpi (76). Colonization observed in our study may represent a combination of external growth during the earliest stages and internal growth via the rachis during 6, 8 and 10dpi. In the future, the extraction and GC ECD methods could be adapted to analyze floral components, single kernels and/or the rachis.
This would provide a more precise picture of fungal colonization of wheat heads and florets.
The objective of this work was to observe the effects of temperature and host on ergosterol and DON production and accumulation. It was hypothesized that lower
102 temperature (15 oC compared to 22 oC) would result in limited fungal growth, but would have little effect on toxin production or translocation. These temperatures were chosen because they are realistic conditions observed during wheat seasons and FHB epidemics in the U.S. (34, 97, 168). Colonization rate was unaffected by temperature, as F. graminearum was isolated from all florets examined by 12dpi, regardless of incubation temperature. However, results show that temperature had significant effects on fungal biomass, as estimated by ergosterol. As expected, wheat incubated at 15 oC contained approximately half the amount of ergosterol as wheat incubated at 22 oC. The warmer temperature favors pathogen germination and proliferation during the early stages of
FHB development. Anderson (1948) found ideal germination conditions to be between
20 and 32 oC for 6 hours. Optimal temperatures for F. graminearum infections are 20
30 oC along with 36 to 60 hours of moisture (6, 168). The direct effects of temperature on ergosterol concentration in F. graminearum mycelium, is unknown and requires further research. An in vitro study suggests that small temperature differences may significantly affect ergosterol and fungal biomass during early development, but have less of an effect over time (4). Future work also includes studying ergosterol accumulation throughout kernel development, until harvest.
The effect of temperature alone on DON was not significant, but the effect of the interaction between temperature and cultivar on DON and ergosterol accumulation was significant. In the susceptible cultivar, more DON was observed in wheat heads incubated at 22 oC (0.134ng/µl) than at 15 oC (0.078ng/µl). ‘Wheaton’ heads contained very similar amounts of ergosterol regardless of the temperature treatments; 0.699ng/µl and 0.721ng/µl in 15 and 22 oC, respectively. This suggests that small temperature differences have little effect on fungal growth in a susceptible cultivar. Cooler temperatures and a susceptible host did not limit fungal growth but slightly limited DON
103 production compared to the warmer temperature. Ergosterol and DON accumulation in
‘Alsen’, the moderately resistant cultivar, appeared to be more affected by temperature than in ‘Wheaton’. There was significantly less fungal biomass in ‘Alsen’ as a result of the 15 oC treatment (0.215ng/µl) than in the 22 oC treatment (0.994ng/µl). ‘Alsen’ incubated at 15 oC had the least ergosterol out of any cultivar temperature combination.
However, the same interaction produced the most DON (0.172ng/µl). A moderately resistant host and cooler temperatures appeared to inhibit pathogen colonization and stimulate mycotoxin production. These results contradict previous studies that found a positive correlation between DON and ergosterol (111) and those that found increased
DON accumulation in susceptible hosts (76). The interaction between environmental conditions, such as temperature, and host genotype may have more powerful effects on
FHB and mycotoxin production than each factor alone.
The present study suggests that growth inhibiting conditions, such as a combination of low temperature and resistant host, may stimulate the fungus to increase
DON production. Several studies indicate that toxigenic fungi respond to stress by increasing mycotoxin production. Schmidt Heydt et al. (2008) studied the effects of
“sub optimal” growth conditions on Fusarium culmorum , another causal agent of FHB.
At high water activity (a w= 0.99), toxin production, estimated by toxin biosynthesis gene expression, appeared to be stimulated by high (>30 oC) and low (<17 oC) temperatures
(155). Trichothecene production was lower at moderate temperatures (20 and 25 oC) which are optimal for fungal growth. At 30 oC, trichothecene production was stimulated by dry conditions (a w= 0.93). Fusarium is not the only mycotoxigenic fungus that responds to stressful conditions with toxin production. In Aspergillus parasiticus , aflatoxin production increased when cultures were incubated at 15 oC. This study also found that Penicillium verrucosum exhibited reduced growth rate as water activity,
104 temperature and pH decreased (155). However, ochratoxin production by P. verrucosum was stimulated by the same conditions which retarded fungal growth in culture.
Biotic factors that cause fungal stress, such as host and other microbe responses, may also contribute to mycotoxin production. Competition between FHB pathogens has been shown to stimulate trichothecene production (186). Xu et al. (2007) inoculated wheat heads with either a single Fusarium species or a combination of F. graminearum,
F. culmorum or F. poae . There was no difference in F. graminearum biomass when inoculated alone or in tandem with another species. However, wheat heads co inoculated with F. graminearum and another species contained 1500 times the DON as in heads inoculated with F. graminearum alone. It was suggested that F. graminearum , the more aggressive species, produced more DON to out compete other species for resources in the wheat heads (186).
A resistant host can also create an unfavorable environment for fungal growth.
An early defense response produced by plants is an oxidative burst, producing reactive oxygen species compounds such as hydrogen peroxide. Ponts et al. (2009) studied the effects of oxidative stress on F. graminearum and F. culmorum. To combat oxidative stress, DON chemotypes, of both species, produced higher levels of toxin in response to
H2O2. Nivalenol chemotypes produced a ‘catalase’ enzyme in response to H 2O2. This enzyme catalyzes the conversion of H 2O2 into water and oxygen gas. Without the threat of free radicals and oxidative stress, the NIV chemotypes did not require high levels of toxin production to survive (135). In the present study, the more resistant cultivar may have created a similar inhospitable environment for F. graminearum which may explain the higher levels of DON observed in ‘Alsen’ than ‘Wheaton’ at 15 oC.
Both cultivars experienced an overall decline in ergosterol (1.519ng/µl to
0.897ng/µl) levels between 8 and 10dpi. The cause for this decline is unknown. While
105 ergosterol decline during FHB infections has been reported (111), this usually occurs as wheat approaches maturity not during early infection stages. It is important to note that fungal colonization of the rachis was not assessed in the present study. By analyzing florets only, fungal biomass present in other portions of the wheat head was not accounted for.
Deoxynivalenol levels also did not increase between 8 and 10dpi. This may be due to the increase in deoxynivalenol 3 glucoside (DON gluc) formation in the wheat heads. In the present study, formation of this conjugated mycotoxin was first detected by
6dpi. ‘Wheaton’, the more susceptible cultivar, appeared to be the more efficient DON conjugator than ‘Alsen’. This suggests that there must be other enzymes able to conjugate DON other that the glucosyltransferase associated with the resistance QTL
FHB1, as ‘Wheaton’ does not contain FHB1 . It has not been confirmed whether DON gluc is transported through the vascular tissue, along with DON. Rachis tissue between florets could be analyzed via the GC ECD method to estimate the total amount and location of mycotoxin and conjugate within wheat heads.
Very little published information exists on the accumulation and occurrence of
DON gluc in wheat. Berthiller et al. (2005) examined naturally contaminated grain and found that 100% of samples tested contained DON gluc. The DON gluc concentration ranged from 4 12% of the DON concentration (18). On the other hand, Sasanya et al
(2008) did not detect DON gluc in every grain sample tested but found DON gluc levels in certain samples to be greater than the DON concentrations. In fact, mean DON levels were 3.4 ± 4.0 µg/g, while mean DON gluc levels were 3.8 ± 8.3 µg/g (148). In the present study, DON gluc was not detected in every floret sample tested, even in those which were positive for DON during 6 to 10dpi. The DON gluc concentration ranged from 9.8 to 332.7% of the DON concentration, when averaged across wheat heads. In
106 three cases the DON gluc concentration was over 200% greater than the DON concentration. These results certainly question the appropriateness of current toxin testing methods used at grain elevators and processors, which do not include DON gluc.
Grain products containing the FDA limit of <1ppm DON could potentially contain additional DON gluc. Ingestion and glucoside cleavage could result in DON levels much greater than the recommended daily tolerable intake. This is of special concern to those with weakened immune systems, poor nutrition, the young or elderly and to the health of sensitive livestock.
Future work also includes, extending harvest dates to monitor fungal growth and deoxynivalenol accumulation throughout wheat development until grain maturity. For wheat producers, grain millers and consumers, it is essential to know the concentration of
DON and DON gluc at harvest and during storage. Additional replications with different cultivars, including winter and spring wheat, may provide more insight into the relationship between Fusarium and trichothecenes during FHB infections. There are still many unknowns regarding mycotoxin synthesis, accumulation and fate in wheat, especially as F. graminearum is subjected to a combination of stressful conditions. Other stress inducing conditions, such as drought or microbial competition, on fungal growth, toxin production and host response could be studied using this system.
107
Chapter 5
SUMMARY A D FUTURE DIRECTIO S
While Fusarium Head Blight (FHB) has been studied for well over a century, questions remain regarding the disease cycle, especially concerning mycotoxin synthesis and accumulation during wheat development. The main objective of this dissertation was to characterize Fusarium graminearum infections and mycotoxin translocation and accumulation in wheat. To better understand these aspects of the FHB disease cycle, a field and growth chamber studies were designed. The field experiment was intended to examine the effects of infection timing relative to host growth stage on FHB symptoms and deoxynivalenol (DON) accumulation in mature grain. Misting chambers and moveable greenhouses were used to facilitate infections during anthesis and/or late milk stages of grain development. The hypotheses were (i) infections during anthesis promote severe symptoms and DON accumulation; (ii) infections during late milk would result in few symptoms and > 2ppm DON; and (iii) the effects of infections during both stages on symptoms and DON would be additive. This study confirmed that infections during both anthesis and late milk contribute to FHB development and DON accumulation.
Infections during anthesis led to greater symptoms and higher levels of DON contamination. Infections during late milk stages alone contributed to DON accumulation (> 2ppm in 2006) yet symptoms in the field and on harvested grain were relatively mild. Increased levels of moisture, as observed in plots which were misted during anthesis and late milk, produced high levels of kernel damage and DON
108 accumulation. The effects of post anthesis moisture have been shown to contribute to
DON loss in grain (33) while other studies have found extended moisture to increase
DON (30). The effects of infections during both growth stages on FHB and DON was not necessarily additive, as symptom development and toxin accumulation are also cultivar specific. Our study differed from previous work, as inoculum and supplemental misting were applied together and during specific growth stages. While infections during anthesis resulted in greater disease and DON levels, this work demonstrated that infections during the grain filling stages also play a role in FHB development and DON accumulation in soft red winter wheat. In the future, additional cultivars could be included in this experiment. This would provide interesting information, as response to infection timing is cultivar specific. The moveable greenhouses provide the means of controlling moisture in a field setting. They could be used to assess the effects of infection timing during other growth stages such as pre anthesis or mid to late dough.
Once infection occurs, questions remain about how F. graminearum colonizes wheat heads and when DON translocation occurs. Previous work has shown that the fungus can enter the rachis within days of floret infection (76). The pathogen is then likely to travel downward from the point of inoculation (7), while DON is able to translocate to areas of the wheat head uncolonized by the fungus (75). The second goal of this thesis was to develop an analytical method which could be used to study the colonization patterns of F. graminearum and mycotoxin translocation in wheat heads. By harvesting wheat florets at scheduled intervals post inoculation and analyzing them for
DON and ergosterol, a fungal biomass indicator, we can uncover infection patterns.
Deoxynivalenol 3 glucoside, a conjugated mycotoxin, is also important for understanding
109 FHB infections. This compound is formed in planta when glycosyltransferases bind a glucoside molecule to DON, rendering it non toxic (96, 137). Existing published protocols were amended to allow for extraction of DON and ergosterol from a single wheat floret (42, 112). Samples were derivatized with hepta –fluorobutyric anhydride and a gas chromatography method was adapted from Croteau et al (1994). Standard curves were developed with limits of detection of 0.005, 0.100 and 0.050 ng/µl for DON,
DON gluc and ergosterol, respectively. This method provides a means of simultaneously detecting trace levels of DON, DON gluc and ergosterol in single wheat florets affected by FHB. This is especially exciting as there is very little information about the frequency and levels of conjugated mycotoxins in food and feed
The third objective was to use the GC method to study FHB infections in two spring wheat cultivars. At mid anthesis a central spikelet was inoculated with F. graminearum macroconidia, and then incubated at 15 or 22 oC. The hypothesis was that cooler temperatures may inhibit fungal colonization and promote DON production.
Spikelets were harvested on 2, 4, 6, 8 and 10 days post inoculation (dpi). One floret from each spikelet was placed on Nash agar to determine F. graminearum incidence. Greater fungal incidence was observed in the susceptible cultivar and was also greater when plants were incubated at 22 oC. Deoxynivalenol translocation to florets not colonized by
Fusarium was observed by 2dpi. The interaction between cultivar and temperature treatment had significant effects on DON and ergosterol accumulation in wheat heads.
‘Alsen’, a moderately resistant cultivar, accumulated the most DON and least ergosterol when incubated at 15 oC. This suggests that host resistance and cool temperatures limited fungal growth yet, had little effect on DON accumulation during early stages of infection.
110 Abiotic factors, such as temperature, have stimulatory effects on mycotoxin production when fungi are under stress (155). Incidence, DON and fungal biomass increased with incubation period post inoculation; however, there was a decline in DON and ergosterol between 8 and 10dpi. We hypothesize that fungal colonization of and toxin translocation to the rachis and the formation of DON gluc during 6 to 10dpi may explain this decline.
Additional research throughout grain development is needed to further characterize the relationship between fungal growth, DON and DON gluc in wheat heads, including analysis of the rachis tissue.
Fusarium graminearum infections, trichothecene production and the epidemiology of FHB are dependent on a multitude of complicated and interacting factors. In general, FHB symptoms positively correlate with DON accumulation in wheat grain (123). However, it is increasingly apparent that combinations of factors, such as moisture, infection timing, temperature and host resistance, which promote FHB severity and symptoms may not be identical with those that stimulate DON accumulation.
Forecasting models for several plant diseases, such as FHB, attempt to make sense of these FHB influencing factors for the benefit of growers and fellow researchers.
However, these models are in need of constant updating and certainly the data presented here could be included in future FHB models, especially a DON forecasting model (86,
114, 150). A better understanding of FHB infections in wheat, as well as DON and
DON gluc accumulation in grain, will ultimately provide information for better management options, thereby improving security and safety for our food and feed supply.
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APPE DIX
ZADOKS ’ SMALL GRAI GROWTH STAGE SYSTEM
Zadoks scale General Description Booting 40 ---- Germination 41 Flag leaf sheath extending 00 Dry seed 45 Boot just swollen 01 Water uptake (imbibition) started 47 Flag leaf sheath opening 03 Imbibition complete 49 First awns visible 05 Radicle emerged from seed 07 Coleoptile emerged from seed Heading 09 Leaf just at coleoptile tip 50 First spikelet of head visible 53 1/4 of head emerged Seedling development 55 1/2 of head emerged 10 First leaf emerged 57 3/4 of head emerged 11 First leaf unfolded 59 Emergence of head complete 12 2 leaves unfolded 13 3 leaves unfolded Flowering or Anthesis 14 4 leaves unfolded 60 Beginning of flowering 15 5 leaves unfolded 65 Flowering half complete 16 6 leaves unfolded 69 Flowering complete 17 7 leaves unfolded 18 8 leaves unfolded Milk 19 9 or more leaves unfolded 70 ---- 71 Kernel watery Tillering 73 Early milk 20 Main shoot only 75 Medium milk 21 Main shoot and 1 tiller 77 Late milk 22 Main shoot and 2 tillers 23 Main shoot and 3 tillers Dough 24 Main shoot and 4 tillers 80 ---- 25 Main shoot and 5 tillers 83 Early dough 26 Main shoot and 6 tillers 85 Soft dough 27 Main shoot and 7 tillers 87 Hard dough 28 Main shoot and 8 tillers 29 Main shoot and 9 or more tillers Ripening 90 ---- Stem elongation or jointing 91 Kernel hard (difficult to 30 Pseudo stem erection separate by fingernail) 31 1st node detectable 92 Kernel hard 32 2nd node detectable 93 Kernel loosening in daytime 33 3rd node detectable 94 Overripe, straw dead and collapsing 34 4th node detectable 95 Seed dormant 35 5th node detectable 96 50% of viable seed germinates 36 6th node detectable 97 Seed not dormant 37 Flag leaf just visible 98 Secondary dormancy 39 Flag leaf ligule/collar just visible 99 Secondary dormancy lost
VITA
Katelyn Tilley Willyerd
210 Buckhout Laboratory, The Pennsylvania State University University Park, PA 16802
Education B.S. in Life Science, with a concentration in Plant Science. (June 2005) Cum laude and with Distinction Otterbein College, Westerville, OH 43081
Certificate in College Teaching. (December 2008) The Schreyer Institute for Teaching Excellence, Penn State.
Honors and Awards I. E. Melhus Award, American Phytopathological Society. (2009) Instruction & Curriculum Committee, Dept. of Plant Path, Penn State. (2008 09) Tag Along Fund, College of Agricultural Sciences, Penn State. (2008) Dept. of Plant Pathology Travel Award, Penn State. (2008) Graduate Student Travel Award, American Phytopathological Society. (2008) College of Agricultural Sciences Student Travel Award, Penn State. (2008) H. W. Popp Scholarship, Penn State. (2007 and 2008) Gamma Sigma Delta (2007 to present) 2nd Place, The Graduate Exhibition, Penn State. (2007) President, Plant Pathology Association, Penn State. (2006 07) Outreach Committee, Dept. of Plant Path, Penn State. (2006 07) C. W. Botts Award, Otterbein College. (2005) Life Science Dept. Award, Otterbein College. (2005)
Professional Membership American Phytopathological Society (2005 to present)
Grants: College of Agricultural Sciences Competitive Grants Program, Penn State. (2008)