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Factors Affecting Fusarium Head Blight Development and Trichothecene Accumulation in Fusarium-infected Wheat Heads

Doctoral Thesis / Dissertation 2010 232 Pages

Biology - Botany

Excerpt

Table of Contents

Acknowledgements

Dedication

Abstract

Table of Contents

List of Tables

List of Figures

Chapter 1: General Introduction

Chapter 2: Literature Review
2.1 History
2.2 Economic importance
2.3 Causal organisms
2.4 Sign and symptoms
2.5 Biology of Fusarium graminearum
2.6 Mycotoxins production
2.7 Impact on grain quality
2.8 Epidemiology
2.9 Genetics of FHB resistance
2.10 FHB management

Chapter 3: Impact of Post-Inoculation Moisture on Fusarium Head Blight (FHB) Development and Trichothecene Accumulation in Spring Wheat
3.1 Introduction
3.2 Materials and methods
3.2.1 Inoculum production
3.2.2 Field experiment design
3.2.3 Inoculation
3.2.4 Disease rating, sampling and VSK analysis
3.2.5 Trichothecene analysis
3.2.6 Data analysis
3.3 Results
3.3.1 Growing season and weather
3.3.2 Grain harvested at maturity
3.3.2.1 Disease Severity
3.3.2.2 Visually scabby kernel (VSK)
3.3.2.3 Deoxynivalenol (DON)
3.3.2.4 15-acetyldeoxynivalenol (15-ADON)
3.3.2.5 3-acetyldeoxynivalenol (3-ADON)
3.3.2.6 Nivalenol (NIV)
3.3.2.7 Correlations
3.3.3 Whole head samples
3.3.3.1 Disease severity
3.3.3.2 Deoxynivalenol (DON)
3.3.3.3 15-acetyldeoxynivalenol (15-ADON)
3.3.3.4 3-acetyldeoxynivalenol (3-ADON)
3.3.3.5 Nivalenol (NIV)
3.3.3.6 Correlations
3.3.4 Whole head samples versus mature harvested grain
3.4 Discussion

Chapter 4: Fusarium Head Blight Development and Trichothecene Accumulaton in Fusarium -Infected Wheat Heads
4.1 Introduction
4.2 Materials and methods
4.2.1 Greenhouse planting
4.2.2 Inoculum preparation
4.2.3 Inoculation disease assessment and sampling
4.2.3.1 Point inoculation
4.2.3.2 Spray inoculation
4.2.4 Trichothecene analysis
4.2.5. Data analysis
4.3 Results
4.3.1. Disease severity
4.3.2. Deoxynivalenol (DON)
4.3.3. 15-acetyldeoxynivalenol (15-ADON)
4.3.4. 3-acetyldeoxynivalenol (3-ADON)
4.3.5. Nivalenol (NIV)
4.3.6. Correlations
4.4 Discussion

Chapter 5: Evaluation of Impact of Single Wetting Event on Trichothecene Accumulation
5.1 Introduction
5.2 Materials and methods
5.3 Results
5.3.1. Disease severity
5.3.2. Deoxynivalenol (DON)
5.3.3. 15-acetyldeoxynivalenol (15-ADON)
5.3.4. 3-acetyldeoxynivalenol (3-ADON)
5.3.5. Mycotoxins in run-off water
5.3.6. Correlations
5.4 Discussion

Chapter 6: General Discussion

References

Appendices

Acknowledgements

I would like to express my heartfelt gratitude to Dr. Ruth Dill-Macky for the opportunity and support towards this degree. Sincere appreciation goes to her for her excellent guidance and advice in this research and other academic area.

My thanks are due to Drs. Brian Steffenson, James Kurle and James A. Anderson for graciously accepting to serve on my doctoral committee and for providing valuable comments and suggestions as and when necessary.

I would like to extend thank to Dr. Yanhong Dong, Mycotoxin laboratory at University of Minnesota for help in mycotoxins analyses. I am thankful to the Small Grain Pathology Lab members; Beheshteh Zargaran and Tracy Scanlan, and former lab members Amar Elakkad and Karen Wennberg, for their valuable assistance in conducting experiments. Without them my research wouldn’t have been completed successfully. My sincere thanks go to Susan Durham, Utah State University and Dr. Aaron Rendahl, University of Minnesota for providing assistance in statistical analysis. Last but not least, I would like to thank the US Wheat and Barley Scab Initiative for funding this research.

Dedication

This dissertation is dedicated to three persons who shaped me to become who I am. First, my late father Revati Raman Gautam, who always dreamed me to be educated to the highest level. Second, my mother Shobha Gautam, who devoted her life to educate her children. Finally, my wife Muna Kadariya, who supported in my each and every endeavor despite a painful separation during my doctoral period.

Abstract

Fusarium head blight (FHB), primarily caused by Fusarium graminearum Schwabe, is an economically important disease as it results in yield loss and quality losses of infected grain and the accumulation of mycotoxins produced by the invading fungus. Environmental factors, host genetics, and isolate aggressiveness impact FHB development and subsequently trichothecene production and accumulation. Though it is well established that moisture around anthesis promotes FHB development and trichothecene accumulation, the role of moisture, either in the form of rainfall or mist-irrigation during the period from anthesis to harvest has been largely overlooked. A three year field experiment was conducted in 2006, 2007 and 2008 to examine the influence of environmental factors, especially moisture, host resistance, and pathogen variation with respect to mycotoxin production capacity and pathogen aggressiveness, on infection, FHB development and mycotoxin production and accumulation in planta. In mature harvested grain FHB severity, visually scabby kernel (VSK) and mycotoxin concentration were significantly higher in Wheaton (FHB susceptible) than in the other two cultivars examined, Alsen and 2375. Although FHB severities were not significantly different in plots receiving different durations of mist-irrigation, VSK were significantly lower in the treatments receiving the least amount of mist-irrigation (14 DAI) than for treatments receiving mist-irrigation for longer periods, suggesting that extended periods of moisture promote disease development. DON concentration in harvested grain was, however, significantly lower in the treatment receiving the longest duration of mist-irrigation than those treatments receiving less water. In the whole head samples, which were collected 0, 7, 11, 14, 21, 28 and 41 days after inoculation, DON and other trichothecenes either declined with increased durations of mist-irrigation or remained low while water was being applied by the misting system. However, trichothecene accumulation was observed to increase after the cessation of mist-irrigation, with increases being most pronounced for the treatments with shorter mist-irrigation periods. The largest reduction in DON observed as a result of extended mist-irrigation periods was seen in the susceptible cultivar Wheaton. The influence of host resistance and pathogen variation on infection, FHB infection, disease development and mycotoxin accumulation in planta was examined in the series of greenhouse experiments utilizing point and spray inoculations. The levels of FHB severity and mycotoxins were higher in spray inoculated experiment than point inoculation in all cultivars examined. Wheaton (FHB susceptible) had the highest FHB severity and levels of mycotoxins. Alsen (moderately resistant to FHB) had significantly lower FHB severities, DON, 15-ADON, 3-ADON and NIV than either 2375 or Wheaton. Though there were no significant differences in initial infection among cultivars examined, Alsen had reduced spread of FHB symptoms from initial infection presumably due to type II resistance. DON production did not peak in all treatments, but where evident, the peak was earlier in 2375 (11 dai) than Alsen and Wheaton (21 or 14 dai). Multiple peaks and declines in DON levels were also evident. The performance of isolates was highly variable, though generally isolates Butte86Ada-11 and B63A were the most aggressive isolates and 49-3 and B45A were the least.

The impact of free moisture, such as that from irrigation systems or rainfall, on mycotoxin accumulation was evaluated in greenhouse experiments. Despite the similar levels of FHB severity observed, the levels of mycotoxins were significantly less in the plants that received a single six hour wetting treatment compared to the respective control. The loss of DON and other mycotoxins was evident in all cultivars examined. Further, DON and 15-ADON were detected in run-off water.

The results of these studies suggest that the availability of free moisture such as from mist-irrigation or rainfall may increase FHB severity and VSK, although DON and other trichothecene concentrations may be concomitantly reduced. Leaching appears to contribute to reductions in DON following wetting events.

List of Tables

Table 3.1. Spearman’s rank correlations of Fusarium head blight (FHB) severity, visually scabby kernels (VSK), deoxynivalenol (DON), and 15-acetyldeoxynivalenol (15-ADON) of mature grain harvested in 2006

Table 3.2. Spearman’s rank correlations of Fusarium head blight (FHB) severity, visually scabby kernels (VSK), deoxynivalenol (DON), 15-acetyldeoxynivalenol (15-ADON), 3-acetyldeoxynivalenol (3-ADON) and nivalenol (NIV) of mature grain harvested in 2007-2008

Table 3.3. Spearman’s rank correlation of Fusarium head blight (FHB) severity, deoxynivalenol (DON), 15-acetyldeoxynivalenol (15-ADON), 3-acetyldeoxynivalenol (3-ADON) and nivalenol (NIV) in whole head samples harvested 7, 11, 14 and 21 dai in 2007-2008

Table 3.4 Spearman’s rank correlation of deoxynivalenol (DON), 15-acetyldeoxynivalenol (15-ADON), 3-acetyldeoxynivalenol (3-ADON) and nivalenol (NIV) in whole head samples harvested 41 dai and harvested mature grain subjected to four mist-irrigation duration treatemtns (14, 21, 28 and 35 DAI) in 2007-2008

Table 4.1. Spearman’s rank correlations for Fusarium head blight (FHB) severity, deoxynivalenol (DON), 15-acetyldeoxynivalenol (15-ADON), 3-acetyldeoxynivalenol (3-ADON) detected in centrally located spikelets of three wheat cultivars point inoculated with five Fusarium graminearum isolates sampled 3, 7, 11, 14, and 21 dai in greenhouse experiments run 1 and 2

Table 4.2. Spearman’s rank correlations for Fusarium head blight (FHB) severity, deoxynivalenol (DON), 15-acetyldeoxynivalenol (15-ADON), 3-acetyldeoxynivalenol (3-ADON) and nivalenol (NIV) detected 7, 11, 14, 21 and 30 dai in the spray inoculated wheat heads in greenhouse experiments run 1 and 2

Table 5.1. Spearman’s rank correlations for FHB severity, deoxynivalenol (DON), 15-acetyldeoxynivalenol (15-ADON) and 3-acetyldeoxynivalenol (3-ADON) detected 7, 14, 21 or 28 dai in control and six hour wetting treatments in greenhouse experiment run 1

Table 5.2. Spearman’s rank correlations for FHB severity, deoxynivalenol (DON), 15-acetyldeoxynivalenol (15-ADON) and 3-acetyldeoxynivalenol (3-ADON) detected 7, 14, 21 or 28 dai in control and six hour wetting treatments in greenhouse experiment run 2

List of Figures

Figure 3.1. Average daily temperature and daily rainfall in 2006-2008

Figure 3.2. Fusarium head blight (FHB) severity (%), percentage visually scabby kernels (VSK), deoxynivalenol (DON, μg g-1 ), and 15-acetyldeoxynivalenol (15-ADON, μg g-1 ) in grain harvested at maturity for three wheat cultivars (Alsen, 2375 and Wheaton) inoculated with five isolates of F. graminearum and a non-inoculated water control in 2006

Figure 3.3. Fusarium head blight (FHB) severities in grain harvested at maturity for three wheat cultivars (Alsen, 2375 and Wheaton) inoculated with five isolates of F. graminearum and a non-inoculated water control in 2007 and 2008

Figure 3.4. Percentage visually scabby kernels (VSK) and deoxynivalenol (DON, μg g-1 ) in grain harvested at maturity for three wheat cultivars (Alsen, 2375 and Wheaton) subjected to four mist irrigation duration treatments (14, 21, 28, 35 DAI) in 2007

Figure 3.5. Percentage visually scabby kernels (VSK) and deoxynivalenol (DON, μg g-1 ) in grain harvested at maturity for three wheat cultivars (Alsen, 2375 and Wheaton) subjected to four mist irrigation duration treatments (14, 21, 28, 35 DAI) in 2008

Figure 3.6. 15-acetyldeoxynivalenol (15-ADON, μg g-1 ) and 3-acetyldeoxynivalenol (3-ADON, μg g-1 ) in grain harvested at maturity of three wheat cultivars (Alsen, 2375 and Wheaton) subjected to four mist irrigation duration treatments (14, 21, 28, 35 DAI) in 2007

Figure 3.7. 15-acetyldeoxynivalenol (15-ADON, μg g-1 ) and 3-acetyldeoxynivalenol (3-ADON, μg g-1 ) in grain harvested at maturity from three wheat cultivars (Alsen, 2375 and Wheaton) subjected to four mist irrigation duration treatments (14, 21, 28, 35 DAI) in 2008

Figure 3.8. Nivalenol (NIV) concentrations (μg g-1 ) in grain harvested at maturity for three wheat cultivars (Alsen, 2375 and Wheaton) subjected to four mist irrigation duration treatments (14, 21, 28, 35 DAI) in 2007 and 2008

Figure 3.9. FHB severity (%) in whole head samples of Alsen, 2375 and Wheaton inoculated with F. graminearum isolates 49-3 and Butte86Ada-11 subjected to mist-irrigation duration treatments 21 DAI in 2007 and 28 DAI in 2008 sampled 0, 7, 11, 14, and 21 dai in 2007 and in 2008

Figure 3.10. Deoxynivalenol (DON, μg g-1 ) in whole head samples of Alsen, 2375 and Wheaton inoculated with F. graminearum isolates 49-3 and Butte86Ada-11 subjected to four mist-irrigation duration (14, 21, 28 and 35 DAI) treatments sampled 7, 11, 14, 21, 28 and 41 dai in 2007 and in 2008

Figure 3.11. 15-acetyldeoxynivalenol (15-ADON, μg g-1 ) in whole head samples of Alsen, 2375 and Wheaton inoculated with F. graminearum isolates 49-3 and Butte86Ada-11 subjected to four mist-irrigation duration (14, 21, 28 and 35 DAI) treatments sampled 7, 11, 14, 21, 28 and 41 dai in 2007 and in 2008

Figure 3.12. 3-acetyldeoxynivalenol (3-ADON, μg g-1 ) in whole head samples of Alsen, 2375 and Wheaton inoculated with F. graminearum isolates 49-3 and Butte86Ada-11 subjected to four mist-irrigation duration (14, 21, 28 and 35 DAI) treatments sampled 7, 11, 14, 21, 28 and 41 dai in 2007 and in 2008

Figure 3.13. Nivalenol (NIV, μg g-1 ) in whole head samples of Alsen, 2375 and Wheaton inoculated with F. graminearum isolates 49-3 and Butte86Ada-11 subjected to four mist-irrigation duration (14, 21, 28 and 35 DAI) treatments sampled 7, 11, 14, 21, 28 and 41 dai in 2007 and 2008

Figure 4.1. Fusarium head blight (FHB) severity (%) observed 0, 3, 7, 11, 14 and 21 dai in spikes of Alsen, 2375 and Wheaton after point inoculation of a centrally located spikelet with five isolates (Butte86Ada-11, 81-2, B45A, B63A and 49-3) of F. graminearum

Figure 4.2. Number of symptomatic spikelets observed 3, 7, 11, 14, 21 and 30 dai in spikes of Alsen, 2375 and Wheaton spray inoculated with five isolates (Butte86Ada-11, 81-2, B45A, B63A and 49-3) of F. graminearum

Figure 4.3. Fusarium head blight (FHB) severity (%) observed 0, 7, 11, 14, 21 and 30 dai in spikes of Alsen, 2375 and Wheaton spray inoculated with five isolates (Butte86Ada-11, 81-2, B45A, B63A and 49-3) of F. graminearum

Figure 4.4. Deoxynivalenol (DON, μg g-1 ) detected 0, 3, 7, 11, 14 and 21 dai in point inoculated centrally located spikelets of Alsen, 2375 and Wheaton inoculated with five isolates (Butte86Ada-11, 81-2, B45A, B63A and 49-3) of F. graminearum

Figure 4.5. Deoxynivalenol (DON, μg g-1 ) detected 0, 7, 11, 14, 21 and 30 dai in kernels from spikes of Alsen, 2375 and Wheaton spray inoculated with five isolates (Butte86Ada-11, 81-2, B45A, B63A and 49-3) of F. graminearum

Figure 4.6. 15-acetyldeoxynivalenol (15-ADON, μg g-1 ) detected 0, 3, 7, 11, 14 and 21 dai in point inoculated centrally located spikelets of Alsen, 2375 and Wheaton inoculated with five isolates (Butte86Ada-11, 81-2, B45A, B63A and 49-3) of F. graminearum

Figure 4.7. 15-acetyldeoxynivalenol (15-ADON, μg g-1 ) detected 0, 7, 11, 14, 21 and 30 dai in kernels from spikes of Alsen, 2375 and Wheaton spray inoculated with five isolates (Butte86Ada-11, 81-2, B45A, B63A and 49-3) of F. graminearum

Figure 4.8. 3-acetyldeoxynivalenol (3-ADON, μg g-1 ) detected 0, 3, 7, 11, 14 and 21 dai in point inoculated centrally located spikelets of Alsen, 2375 and Wheaton inoculated with five isolates (Butte86Ada-11, 81-2, B45A, B63A and 49-3) of F. graminearum

Figure 4.9. 3-acetyldeoxynivalenol (3-ADON, μg g-1 ) detected 0, 7, 11, 14, 21 and 30 dai in kernels from spikes of Alsen, 2375 and Wheaton spray inoculated with five isolates (Butte86Ada-11, 81-2, B45A, B63A and 49-3) of F. graminearum

Figure 4.10. Nivalenol (NIV, μg g-1 ) detected 0, 7, 11, 14, 21 and 30 dai in kernels from spikes of Alsen 2375 and Wheaton spray inoculated with five isolates (Butte86Ada-11, 81-2, B45A, B63A and 49-3) of F. graminearum

Figure 5.1. Deoxynivalenol (DON, μg g-1 ) detected 7, 14, 21 and 28 dai in Alsen, 2375 and Wheaton inoculated with isolates Butte86Ada-11 and 81-2 of F. graminearum for control plants and plants subjected to a six hour wetting treatment

Figure 5.3. 15-acetyldeoxynivalenol (15-ADON, μg g-1 ) detected 7, 14, 21 and 28 dai in Alsen, 2375 and Wheaton inoculated with isolates Butte86Ada-11 and 81-2 of F. graminearum for control plants and plants subjected to a six hour wetting treatment

Figure 5.4. 3-acetyldeoxynivalenol (3-ADON, μg g-1 ) detected 7, 14, 21 and 28 dai in Alsen, 2375 and Wheaton inoculated with isolates Butte86Ada-11 and 81-2 of F. graminearum for control plants and plants subjected to a six hour wetting treatment

Figure 5.5. Deoxynivalenol (DON, μg per 50 ml of water) detected in run-off water from Alsen, 2375 and Wheaton inoculated with isolates of F. graminearum at anthesis and subjected to a six hour wetting event at 7, 14, 21 or 28 dai

Chapter 1 General Introduction

Fusarium head blight (FHB or scab), a major disease of wheat and other small grains, is incited by several species of Fusarium (Atanasoff, 1920; Parry et al., 1995; Stack and McMullen, 1985). Fusarium graminearum Schwabe [teleomorph Gibberella zeae (Schwein) Petch], F. culmorum W. G. Smith (Sacc.) and F. avenaceum (Fr.) Sacc. (teleomorph G. avenacea R. J. Cooke), however, are the most common causal agents (Parry et al., 1995). In North America, F. graminearum is the primary species causing FHB (McInnes and Fogelman, 1932; Wilcoxson et al., 1988). Temperature appears to be a critical criterion for the distribution of the major Fusarium species. While F. graminearum is more common in warmer and humid regions of the world, including North America, Australia and central Europe and southern Europe (Mesterházy, 2003; Parry et al., 1995), F. culmorum is more frequently isolated in cooler and humid areas including north-western Europe (Parry et al., 1995; Snijders et al., 1990b). Though FHB has not been considered a widespread disease (Parry et al., 1995), in the last two decades it has re-emerged worldwide as a disease of importance (Windels, 2000). Because of its multifaceted effects on crops (Atanasoff, 1920) it has been estimated to have caused economic losses of $2.7 billion in the northern Great Plains and central US from 1998 to 2000 (Nganje et al., 2002). Although FHB has been common, severe and well documented in the past, recent epidemics have resulted in increased concern, greater public interest and expanded research efforts.

Warm temperatures and extended periods of moisture on plant surfaces around anthesis are essential for the infection and colonization of wheat tissues by F. graminearum and the first symptoms of water soaked lesions appear within 2-4 days after infection (Atanasoff, 1920, Xu, 2003). Kernels from infected spikelets are generally smaller and lighter having a shriveled and chalky appearance referred to as ‘tombstones’ or scabby kernels (Abramson et al., 1987; Dickson and Mains, 1929; Johnson and Dickson, 1921; Parry et al., 1995). Formation of tombstones reduces the yield significantly and Fusarium damaged grain tends to exhibit weaker dough properties and unsatisfactory baking quality, making the marketing and processing of the grain difficult (Bechtel et al., 1985; Dexter et al., 1996; Dexter et al., 1997; Wang et al., 2005). In addition to yield losses, the quality of infected grain is compromised due to the production of a range of mycotoxins, including deoxynivalenol (DON), 15-acetyldeoxynivalenol (15-ADON), 3- acetyldeoxynivalenol (3-ADON), and nivalenol (NIV), by the FHB pathogens (Nasri et al., 2006; Pirgozliev et al., 2003). These mycotoxins are hazardous to humans and animals, thus making highly infected grain unfit for food or feed. Wheat from US production fields infected by Fusarium have routinely tested positive for most of these mycotoxins (Abramson et al., 1987; McMullen et al., 1997). DON is considered the most important toxin produced by F. graminearum and is regulated in the grain and wheat based food products by US federal agencies.

FHB resistant wheat is reported to be associated with lower levels of DON contamination than FHB susceptible wheat (Mesterházy et al., 2003; Miller et al., 1985; Wilde and Miedaner, 2006). Whether the reduced DON in cultivars expressing FHB resistance is due to the resistance of the host to initial infection (type I resistance, Schroeder and Christensen, 1963), spread of the fungus in the spike (type II resistance, Schroeder and Christensen, 1963) or modes of resistance either preventing DON synthesis or promoting the degradation of DON (Miller et al., 1985; Miller and Arnison, 1986) has not been clearly established. To facilitate the evaluation and selection of resistant germplasm and to assess commercial crop quality, researchers frequently utilize correlations between visual FHB severity assessments in the field and DON concentrations to predict DON in harvested grain (Arsenuik et al., 1999; Groth et al., 1999; Jones and Mirocha, 1999). Although correlations are generally significant, correlation coefficients are frequently low and vary greatly among locations and years. The reasons for this variability are not fully understood, but may be associated with toxin production capacity of prevalent strains within the Fusarium population, the type and level of host plant resistance to FHB and/or the environmental conditions prevailing between the time of initial infection and harvest.

Environmental factors, primarily moisture, have been reported to have an important influence on toxin production (Hope et al., 2005). Moisture in the form of rainfall or relative humidity, during anthesis or shortly thereafter has been linked to higher FHB incidences, higher FHB severities and DON accumulation (Abramson et al., 1987; Atanasoff, 1920; Rohácik and Hudec, 2005; Tuite et al., 1990). However, the specific environmental factors triggering, regulating or influencing mycotoxin synthesis and accumulation in the infected host are not well understood, (Mesterházy, 1999).

The application of extended supplemental moisture between anthesis and the time of disease rating, generally around 21 days after inoculation (dai), is common in field nurseries screening wheat germplasm for resistance to FHB. Supplemental moisture is generally provided in the form of mist-irrigation. The possible impact of supplemental moisture and rainfall occurring after disease rating on the accumulation of Fusarium -mycotoxins has been largely ignored, however. It has been reported that continuous mist-irrigation for 3 days, after inoculation with F. culmorum, increased DON in harvested wheat compared to either of the overnight or one 2 full day misting (Lacey et al., 1999). Lemmens et al. (2004) used two levels of mist-irrigation treatments (no irrigation and irrigation for 26 days after flowering) and reported a decrease in the DON level in some wheat lines while observing an increase in others. Culler et al. (2007) also reported relatively lower levels of DON with greater environmental moisture, especially in the susceptible hard red spring wheat cultivar, Wheaton. They observed generally lower DON concentrations under misting treatments which ran for 31-32 days after inoculation compared to misting treatments for 15-16 days after inoculation. The differences in DON levels were, however, not significant at all sampling times. In contrast, Cowger et al. (2009) reported increases in FHB severities, Fusarium damaged kernels (FDK) and DON levels with the increased durations of mist-irrigation from 0 to 30 days post-anthesis.

Several studies have reported a positive correlation between isolate aggressiveness and DON production by F. graminearum and F. culmorum (Gang et al., 1998; Hestbjerg et al., 2002; Mesterházy, 2002). However, DON is not essential for pathogenesis (Dyer et al., 2005; Eudes et al., 2001; Proctor et al., 1995). The time from inoculation until the initiation of production of DON reported for wheat varies from 26 hrs (Chen et al., 1996) to 4 days (Savard et al., 2000), which supports findings that DON is not required for initial infection and colonization.

The first objective of this study was to examine the influence of environmental factors, especially moisture, host resistance types, and pathogen variation with respect to mycotoxin production capacity and pathogen aggressiveness, on infection, FHB development and mycotoxin production and accumulation in planta. The second objective of this study was to examine the impact of free environmental moisture, such as from rainfall or simulated by irrigation systems on disease development and mycotoxin production and accumulation in planta. Specifically, the second objective aimed to determine if a single wetting event could result in the leaching of mycotoxins produced in planta from head tissues.

Chapter 2 Literature Review

2.1 History

Fusarium head blight (FHB) of wheat and other small grains usually occurs in warm and humid cereal growing regions of the world (Schroeder and Christensen, 1963; Wilcoxson et al., 1992). The disease was first described by W. G. Smith in England in 1884 (McInnes and Fogelman, 1932). In North America, it was first reported by J. C. Arthur (Arthur, 1891). Fusarium head blight was reported in Ohio, Delaware, Indiana, Iowa, Pennsylvania, and Nebraska before 1900 (Stack, 2003). In Minnesota, the earliest FHB outbreaks reported were in 1905, 1907 and 1915 (MacInnes and Fogelman, 1923). Most of the Midwestern states were also affected by epidemics in 1917 among which Illinois, Indiana and Ohio were hardest hit. By 1919 the disease had been detected in 31 states, with its distribution covering most of the central and eastern states (Atanasoff, 1920). A major outbreak of FHB occurred in 1919 with the greatest loss of wheat reported in Illinois and Iowa (Dickson and Mains, 1929; Stack, 2003). In this epidemic Minnesota recorded losses in wheat production over 10% (Johnson and Dickson, 1921). In 1925 and 1928 outbreaks of FHB were again severe in Minnesota. Barley was heavily affected in a 1928 outbreak (Dickson and Mains, 1929). In the tri-state region of Minnesota, North Dakota and South Dakota, FHB was severe in the southern and eastern parts in 1932 and barley crops in Minnesota and Iowa were affected (Christensen and Stakman, 1935). Outbreaks continued across the country in 1940’s and 1950’s. Minnesota had serious outbreaks of FHB in 1939, 1941, 1944 and 1945 (Hanson et al., 1950). During the 1970’s and 1980’s, FHB epidemics were sporadic (Halfon-Meiri et al., 1979; Tuite et al., 1990), although the disease appeared in eastern and western Canada, and several states of the US including Minnesota (Sutton, 1982; Stack and McMullen, 1985; Wilcoxson et al., 1988). Epidemics which occurred during the 1990’s, mostly across the eastern half of the US, are of prime importance in the history of the FHB. In 1993, Minnesota, North Dakota, South Dakota and the Canadian province of Manitoba were devastated by FHB epidemics. According to McMullen et al. (1997) the 1993 epidemic caused the greatest loss in any single year due to plant disease in the tri-state area and thus rivals the stem rust epidemics that ravaged the Upper Midwest in the early 1900’s (Eversmeyer and Kramer, 2000). A severe epidemic occurred in 1996 on soft red and soft white winter wheat in Iowa, Arkansas, Louisiana, Ohio, Indiana, Illinois, Wisconsin, Michigan, New York and the Canadian province of Ontario (McMullen et al., 1997). Several soft red wheat producing states east of Mississippi River were ravaged by FHB in 2003 (USBWSI, 2004). In 2007, FHB disease was generally low across the US. However, isolated disease outbreaks were serious in Nebraska and Kansas (USBWSI, 2007b). The situation repeated in 2008 with Nebraska and Kansas being hardest hit while much of the rest of the US reported little FHB (USBWSI, 2008).

2.2 Economic importance

Fusarium head blight occurs almost every year but is generally limited to relatively few wheat and barley crops. Thus FHB is not recorded as a widespread disease (Parry et al., 1995). In recent years, however, FHB has re-emerged worldwide as a disease of economic importance (Windels, 2000) with enormous economic impact because of its multifaceted effects on crops (Atanasoff, 1920). The impact of FHB starts right after germination as Fusarium infection of seed can result in reduced germination and post emergence seedling blight (Bechtel et al., 1985; Jones, 1999). However, FHB cannot be seed transmitted through Fusarium -infected seeds (Jones, 1999). In addition to yield losses caused by the FHB, the presence of mycotoxins in infected grain further exacerbates disease losses (McMullen et al, 1997). In the 1917 disease outbreak, wheat yield loss was 288.8 megagram (Mg) and was attributed to several species of Fusarium (Atanasoff, 1920). In the 1928 epidemics, there were yield losses of 20% and 15% in barley and wheat, respectively (Stack, 2003). Yield losses in wheat due to the FHB epidemics during 1990’s in the US was over 18.4 Mg valued at ca. $2.5 billion. Similarly, barley producers lost $400 million in the same time (Windels, 2000). The epidemics of 1990’s in the tri-state area were so serious that there was a net loss in revenue per harvested acre of wheat in the Red River Valley area of North Dakota and Minnesota every year from 1993 to 1998 with exception of Minnesota in 1996 (Windels, 2000). Estimated direct and secondary economic losses by FHB in wheat and barley in the northern Great Plains and central US was $2,679 million from 1998 to 2000 (Nganje et al., 2002). As a consequence of these losses to FHB, land planted to barley from 1991 to 1999, decreased by 77%, 53% and 84% in Minnesota, North Dakota and South Dakota, respectively. Similarly, the area planted to wheat decreased by 6%, 5% and 7% in Minnesota, North Dakota and South Dakota, respectively (NASS, 2009). Many farmers abandoned farming as an occupation and wheat crops became rotational crops and the barley crop was almost wiped out from Minnesota (McMullen, 2003). The decrease in wheat and barley planting from 1991 to 1999 can be attributed primarily due to yield losses caused by FHB and associated quality losses due to mycotoxin accumulation in the infected grain (Windels, 2000).

2.3 Causal organisms

Head blight disease is caused by several species of the fungal genus Fusarium (Atanasoff, 1920; Parry et al. 1995; Stack and McMullen, 1985). Fusarium species that are reported to cause head blight to various extent in cereals are F. acuminatum Ellis & Kellerm. (teleomorph G. acuminata Wollenw.), F. avenaceum (Fr.) Sacc. (teleomorph G. avenacea R.J. Cooke), F. crookwellense Burgess, Nelson & Toussoun, F. culmorum W.G. Smith (Sacc.), F. dimerum Penzig, F. equiseti (Cda.) Sacc., F. equiseti v. bullatum (Sherb.) Wollenw. (teleomorph G. intricans Wollenw.), F. graminearum Schwabe [teleomorph Gibberella zeae (Schwein) Petch], F. lateritium Nees (teleomorph G. baccata (Wallr.) Sacc.), F. merismoides Corda, F. moniliforme Sheldon (synonyms F. verticillioides (Sacc.) Nirenberg) (teleomorph G. moniliformis Wineland), F. orthoceras v. longius (Sherb.) Wollenw., F. oxysporum v. aurantiacum (Lk.) Wollenw., F. poae (Peck) Wollenw., F. proliferatum (Matsush.) Nirenberg ex Gerlach &Nirenberg, F. redolens Wollenw., F. sambucinum Fuckel (teleomorph G. pulicaris), F. scirpi Lamb. Et Fautr., F. semitectum Berk. & Ravelnel, F. solani (Mart. pr. p) App. et Wollenw., F. sporotrichioides Sherb, F. subglutinans (Wollenw. & Reinking) Nelson, Toussoun & Marasas, F. tricinctum (Corda) Sacc., Microdochium nivale (Fries) Samuels & Hallet [synonym Fusarium nivale (Fr.) Ces.] (teleomorph Monographella nivalis) (Abramson et al. 1987; Atanasoff, 1920; Duthie et al., 1986; Liddell, 2003; Parry et al., 1995; Schroeder and Christensen, 1963; Stack, 2003; Stack and McMullen, 1985; Tuite et al., 1990; Vargo et al., 1981; Wilcoxson et al., 1988; Wong et al. 1992).

Though FHB can be incited by several species of Fusarium, F. graminearum, F. culmorum and F. avenaceum are the most common causal agents (Parry et al., 1995). In North America, especially in Minnesota, F. graminearum is the primary species causing head blight (McInnes and Fogelman, 1932; Wilcoxson et al., 1988). Temperature is a critical factor for the distribution of the primary causal agents among the major Fusarium species. While F. graminearum is more common in warmer and humid regions of the world, including North America, Australia and central Europe and southern Europe (Mesterházy, 2003; Parry et al., 1995), F. culmorum dominates in cooler and humid areas including north-western Europe (Parry et al., 1995; Snijders et al., 1990b). Of several species isolated from infected wheat heads in North Dakota, F. graminearum was present in 68% (Stack and McMullen, 1985). Similarly, F. graminearum was isolated from 75% of the heads along with 15 other species isolated from heads collected in farm fields and experimental plots throughout Minnesota (Wilcoxson, 1988). When Tuite et al. (1990) analyzed FHB infected winter wheat samples from Indiana, 96% were infected with F. graminearum. However, when the FHB severity is low, other species are reported to become dominant (Shaner, 2003).

2.4 Sign and symptoms

The symptoms of FHB are similar in all the affected cereals (Dickson and Mains, 1929; Parry et al., 1995). The first symptoms, water-soaked lesions of 2-3 mm in length, appear (Atanasoff, 1920) within 2-4 days after infection under favorable conditions, mostly at the base of the middle spikelets in the middle of the head (Stack, 2003). In water-soaked lesions in the glumes, the veins have a darker olive-green color than the area between veins (Atanasoff, 1920). Soon after the water soaking appears, symptoms spread to the rachis. Through the rachis the fungus can rapidly spread up, down and horizontally in the spike (Parry et al., 1995; Wiese, 1987). Frequently salmon to pink colored fungal growth and orange colored sporodochia can be seen at the base of the spikelets or along the edge of glumes (Arthur, 1891; Johnson and Dickson, 1921, Parry et al 1995). Under humid conditions water-soaked lesions may turn brown to purplish-brown with or without a bleached center (Bennett, 1931). Similarly, early infected spikelets give a ‘scabbed’ appearance due to formation of blue-black perithecia under prolonged moist and humid conditions (Mathre, 1982). If the environment is dry, the water-soaked symptoms turn the typical color of ripe head (Atanasoff, 1920). In most of the cases in susceptible cultivars of wheat, fungal growth in the rachis causes vascular occlusion cutting off the nutrient and water supply to spikelets above the point of infection (Atanasoff, 1921; Bai and Shaner, 1996). This results in healthy spikelets above the infection point drying out and turning to the color of mature heads, (Johnson and Dickson, 1921; Stack, 2003). Such dried spikelets shrink and appress to the rachis. Grains do not form or do not develop fully on such spikelets depending upon the stages of grain at which vascular tissues become dysfunctional. This phenomenon is more evident in susceptible cultivars of wheat (Bai and Shaner, 1996). In some cases one or a few vascular bundles remain uninfected continuing the nutrient supply to the spikelets above the infected spikelets allowing the formation of normal grain (Atanasoff, 1920). In addition to the floret, characteristic signs and symptoms of FHB also develop in kernels. Symptom development in kernel depends on the time of infection. In severely infected and early infected spikelets, kernels do not develop. If kernels develop, they are smaller and light weight with a shriveled and chalky appearance and are referred to ‘tombstones’ or ‘Fusarium damaged kernels’ (FDK) or scabby kernels (Abramson, 1987; Dickson and Mains, 1929; Johnson and Dickson, 1921; Parry et al., 1995). If the infection occurs towards late stage of the kernel development, kernels may achieve normal size and weight, but may look discolored with pinkish areas (Atanasoff, 1920).

2.5 Biology of Fusarium graminearum

F. graminearum belongs to the fungal phylum Ascomycota (Webster and Weber, 2007). The teleomorph or sexual stage of F. graminearum is Gibberella zeae (Schwein) Petch [syns. G. roseum ] (Booth, 1971). G. zeae produces dark purple perithecia, which appear black. Perithecia are generally 140-250 μm in diameter and contain narrowly clavate and thin walled asci. The asci bear eight ascospores which are 3 or more septate, 20-29 μm in length and 3.5-4.5μm in width (Samuels et al., 2001; Webster and Weber, 2007). Each perithecium may bear up to 45,000 ascospores (Khonga and Sutton, 1988).

Burgess et al. (1975) described two groups of F. graminearum; group 1 and group 2. Group 1 isolates were described as homothallic and unable to produce perithecia on potato dextrose agar (PDA) and carnation leaf agar (CLA) media although fertile perithecia have been reported to occur in group 1 (Burgess et al., 1975). Group 2 isolates were described as homothallic and readily able to form fertile perithecia on PDA and CLA media (Burgess et al., 1975; Francis and Burgess, 1977). Group 1 isolates were associated with crown rot of wheat and group 2 isolates were primarily associated with stalk rot of maize and head blight of wheat (Burgess et al., 1975; Purss, 1971). It was suggested that F. graminearum group 1 evolved as soil borne pathogens and group 2 as air borne pathogens (Sutton, 1982). Later Aoki and O’Donnell (1999) reclassified group 1 isolates as F. pseudograminearum (teleomorph G. coronicola Aoki and O’Donnell) based on phylogeny analysis of DNA sequence of -tubulin introns and exons from both groups 1 and 2. Further it was suggested that the morphology of macroconidia can be used reliably to distinguish these two species. While the conidium of F. graminearum is widest at upper region, conidium of F. pseudograminearum is widest at the midregion of their length (Aoki and O’Donnell, 1999; Francis and Burgess, 1977).

When F. graminearum is grown in PDA media, white mycelial colonies grow fast and develop a characteristic color of pink to dark purple (Samuels et al., 2001). F. graminearum produce classic foot-cells and banana shaped, 3-5 septate macroconidia on (Liddel, 2003; Seifert, 2001). Unlike some of other Fusarium, F. graminearum do not produce microconidia (Burgess et al., 1994; Liddel, 2003; Nelson et al., 1983). The size of macroconidia ranges from 41 to 60 μm in length and 4.5 to 5 μm in width (Samuels et al., 2001). Often F. graminearum produce chlamydospores, a thick walled structure formed from macroconidia and/or mycelium, when they are in contact with soil (Sitton and Cook, 1981). Chlamydospores are globose to oval in shape, 8- 12 μm in diameter and pale brown in color. They are generally produced in pairs or short chains (Gerlach et al., 1982). Though the function of chlamydospores in other microorganisms is for survival, its function in F. graminearum is largely unknown.

Conidia, ascospores, chlamydospores and hyphal fragments may serve as inoculum for infection (Dill-Macky, 2003). Ascospores are the principle inoculum in natural conditions. The infection effectiveness is not significantly different for ascospores and conidia (Stack, 1989; Scholz and Steffenson, 2001), thus the use of macroconidia is comparable to ascospores in experiments. Most experiments conducted with F. graminearum use conidia as inoculum because of their hydrophilic nature, ease of production, the ability to quantify inoculum and because they are economic to produce with respect to labor and financial resource (Dill-Macky, 2003). Hyphal fragments cannot be quantified thus its use as inoculum is limited.

Ascospores are forcibly ejected from asci and perithecia (Webster and Weber, 2007). Once they are ejected, they are dispersed by wind (Sutton, 1982). Ascospores have a diurnal discharge pattern which peaks from 1800 to 2400 hours (Paulitz, 1996). The discharge of ascospores is dependent on environmental factors, primarily moisture and temperature. It has been hypothesized that humidity promotes ascospore release through the mechanical rupture of the ascus due to swelling of mucilage around ascospores. However, Tschanz (1975) suggested that drying of perithecia and the substrate on which perithecia form, is required for the forcible ejection of ascospores and maximum ascospores discharge occurs at 16ºC. Though the moisture in the form of humidity or rainfall is required for perithecia development, it is not required for ascospore release. Paulitz (1996) showed that rainfall of >5mm during the day or relative humidity of >80% during the day, suppressed the ascospores ejection. However, ascospores will ooze into the gelatinous matrix around the ostiole, the opening of perithecia, when moisture is prevalent.

In contrast to ascospores, conidia are primarily dispersed by rain splash (Sutton, 1982), thus the distance of their dispersal is limited. Intense rainfall and large raindrops are required to splash macroconidia 70-90 cm above the ground surface to infect wheat head (Madden, 1992). Therefore, macroconidia are generally not splashed in one event from plant residues on the ground to the wheat head (Jenkinson and Parry, 1994). However, spores have been recorded to be splashed up to 100 cm (Paul et al., 2004). While macroconidia are also produced in infected heads later in the season, secondary inoculum has less importance in disease development (Fernando et al., 1997).

2.6 Mycotoxins production

The major concern from FHB, in addition to yield loss, is the production of mycotoxins by Fusarium in infected grain. Mycotoxins are secondary metabolites that are not essential for survival of the fungus (Desjardins et al, 1993). They are toxic against many different organisms, thus called mycotoxins (Webster and Weber, 2007). Different species of Fusarium produce trichothecene mycotoxins (type A or type B) and the estrogenic mycotoxin zeralenone (ZEN). Type A trichothecene include, T-2, H-2, and diacetoxyscirpenol (DAS); and type B trichothecenes include deoxynivalenol (DON), nivalenol (NIV), 3-acetyldeoxynivalenol (3- ADON), 15-acetyldeoxynivalenol (15-ADON) and fusarenon-X (FUS-X) (Nasri et al., 2006; Pirgozliev et al., 2003). North American strains of F. graminearum produce 15-ADON and ZEN, in addition to DON. Most Japanese and Australian strains produce NIV, FUS-X, and ZEN or DON, 3-ADON, and ZEN (Miller et al., 1991). In addition to above mentioned toxins, F. graminearum has been reported to produce other secondary metabolites including 4-acetamido-2- butenoic acid, butenolide, diacetodeoxynivalenol, 3,15-dihydroxy-12,13-epoxytrichothec-9-ene- 8-one, monoacetoxyscirpenol, and neosolaniol (Marasas et al., 1984). Wheat and barley from US production fields infected by Fusarium have routinely tested positive for most of these mycotoxins (Abramson et al., 1987; McMullen et al., 1997). Deoxynivalenol is, however, considered the most important toxin produced by F. graminearum, though it is less toxic than other trichothecene. The DON concentration is regulated in grain and wheat based food products by US federal agencies. The US, Canada and several European countries have set 2 mg kg-1 as the acceptable limit of DON in wheat grain for human consumption (Bai et al., 2001).

Mycotoxins are hazardous to animals, thus making highly infected grain unfit for food or feed. There is long history of mycotoxosis due the consumption of moldy grain by humans and animals. One of the most notorious cases was the outbreak of alimentary toxic aleukia (ATA), in humans, in Russia during 1932. Symptoms included weakness, vertigo, nausea, vomiting, and diarrhea in milder cases to severe skin rashes and necrotic lesions of the gastrointestinal tract in severe cases, often resulting in death (Dounin, 1926; Joffe, 1986). Though the disease and cause was mysterious during the outbreak, after two decades it was identified that the trichothecene T-2 produced by F. sporotrichioides was associated with ATA (Desjardins, 2006). Similarly, outbreaks of human toxicosis during 1890, 1901, 1914, 1920 and 1923 in Japan were associated with grains contaminated with the red mold disease, akakabi-byo. Symptoms included nausea, vomiting, diarrhea, headache, dizziness and visual hallucinations. However, it was not fatal like ATA. Though there is no published proof, it was suspected that DON and NIV from F. graminearum and other Fusarium species were associated with the toxicosis (Marasas et al., 1984).

Feed refusal by swine in the USA in 1928 was the first incident that was linked to F. graminearum and the toxin DON. In 1928 wheat, barley and oats grains were unsellable due to the FHB epidemic. Fusarium -infected grain was used as animal feed. It was reported that swine fed with infected grain became sick with vomiting and refused to eat. Thus DON became known as ‘the refusal factor’ or ‘vomitoxin’ (Desjardins, 2006). Later the effect of DON was proved experimentally by Christensen and Kernkamp (1936). They showed that as little as 13 g of heavily FHB infected barley could induce vomiting in a 132 pound pig. In addition to animals and human, Fusarium toxins are phytotoxic. The phytotoxicity of mycotoxins produced by several species of Fusarium has been demonstrated in pepper, corn, tomato, banana, wheat and several other crops (Bruins et al., 1993; Joffe, 1986; Shimada and Otani, 1990; Wang and Miller, 1988). However, the toxicity among trichothecene differ more than 1000 fold (Rotter et al., 1996). The phytotoxicity of DON results in the inhibition of seed germination and growth of wheat seedlings (Wakulinski, 1989) and the toxin has an inhibitory effect on mitosis (Packa, 1991). The mode of action of trichothecene, including DON, is to inhibit ribosomal protein synthesis in plants. The double bond at C9-C10 and presence of epoxy ring at C-12, 13 in DON is required for its inhibitory action. Inhibition of protein synthesis is achieved through the binding of DON to the 60S subunit of eukaryotic ribosomes and interference with the activity of peptidyltransferase (Ehrlich and Daigle, 1987).

The production and accumulation of DON in infected wheat heads has been shown to be varied after infection until harvest. When Evans et al. (2000) inoculated barley heads with F. graminearum macroconidia, DON was detected as early as 36 hrs after infection. The same study has indicated that DON can be produced in large amounts at 72 hours of infection, being especially apparent following the appearance of necrotic symptoms. Others have reported start of DON production from 26 hours after infection (Chen et al., 1996) up till 4 days (Savard et al., 2000). Miller and Young (1985) reported the start of the production and accumulation of DON in infected heads at about three days after infection, DON increased until six weeks and declined thereafter before it reached a constant level before grain maturity. A similar decline in DON level prior to harvest has been reported in barley (Prom et al., 1999) and from naturally infected wheat fields (Scott, 1984). Some studies have indicated the decline of DON started earlier than six weeks (Argyris et al., 2003; Culler et al, 2007).

Based on gold labeling of an antibody against DON, Kang and Buchenauer (1999) suggested that DON is synthesized in metabolically active areas of fungal hyphae and that the mitochondrion is the organelle of toxin production. DON can be translocated from the site of production to nearby tissues with no fungal invasion due to its water solubility (Desjardins, 2006). Snijders and Krechting (1992) found DON in kernels without fungal invasion. They suggested that the DON was translocated from chaff invaded by fungal hyphae. DON can be transported acropetally and basipetally from the site of infection facilitated by fungal growth and translocation through xylem and phloem (Kang and Buchenauer, 1999). However, accumulation of DON is usually higher in spikelets, rachis and rachilla below the point of infection than above (Savard et al., 2000). Among the different parts of the head, the rachis tends to have the highest concentration of DON (Savard et al., 2000). It might be the case that the rachis is heavily contaminated because, fungal hyphae and DON spread to adjacent spikelets through the rachis and rachilla.

As DON is phytotoxic, it is expected that it might have role in pathogen aggressiveness. The role of DON in pathogen aggressiveness has not been well defined. A close relationship has been demonstrated between toxin production and pathogenic changes in different tissues, symptom appearance and the colonization of the pathogen in the wheat spike (Kang and Buchenauer, 1999). While some studies have reported a positive correlation between aggressiveness and DON production by F. graminearum and F. culmorum (Gang et al., 1998; Hestbjerg et al., 2002; Mesterházy, 2002), others suggest no correlation or an inconsistent correlation (Adam & Hart, 1989). A rapid increase of the disease has reported to occur due to the inhibition of enzymatic activity by DON in susceptible hosts (Snijders, 1994). Several studies have indicated that though the DON is involved in Fusarium aggressiveness, it is not essential to pathogenesis (Desjardins et al., 1996; Dyer et al., 2005; Eudes et al., 2001). Variation in the time till the initiation of production of DON suggests that DON might not be required for initial infection and colonization. The role of mycotoxins in the virulence of F. graminearum on wheat was examined by Proctor et al. (1995) utilizing trichothecene deficient mutants induced by disruption of TRI5 which encodes a trichodiene synthase, the first enzyme in the trichothecene biosynthesis pathway. They observed that the mutant was still pathogenic to wheat but less aggressive than the wild-type. A similar result was observed when the TRI14, a gene with putative function of positive regulator of DON synthesis and possible role in the export of DON outside of mycelia, was mutated (Dyer et al., 2005). DON producers are reported to be twice as aggressive as NIV producing F. graminearum strains (Cumagun et al., 2004). Similar results 12 were obtained when chemotypes and virulence of 246 strains of F. graminearum from Nepal was analyzed (Desjardins et al., 2004).

The pathogenic variability of F. graminearum has been well known since 1929 (Tu, 1929) and confirmed in subsequent studies (Akinsanmi et al., 2006; Gang et al., 1998; Goswami and Kistler, 2005; Xue et al., 2004). Variability for trichothecene production exists naturally in F. graminearum populations (Burgess et al., 1996). Tóth et al. (2005) observed wide variation in DON production (54 - 11471 mg kg-1 ) within different isolates of F. graminearum when inoculated into sterilized rice medium. Almost all isolates they tested were pathogenic despite high variability in aggressiveness. Similar results were observed when 66 isolates of F. graminearum associated with FHB from North Carolina were analyzed for in vitro DON production and pathogenicity on three cultivars of soft red winter wheat (Walker et al., 2001).

2.7 Impact on grain quality

In addition to yield losses and mycotoxin contamination, Fusarium infection causes several chemical compositional changes in the wheat grain impacting desired dough and baking quality. Bechtel et al. (1985) indicated that Fusarium infection of wheat grain results into the enzymatic degradation of starch granules, storage proteins, and cell walls. Fusarium produces proteolytic enzymes, which are required for its successful colonization and utilization of nitrogen and carbon sources (Wang et al., 2005). Proteolytic enzymes hydrolyze endosperm proteins during dough mixing and fermentation and results in weaker dough, decreased loaf volume and unsatisfactory bread quality (Dexter et al., 1996; Dexter et al., 1997, Nightingale et al., 1999). Fusarium infected grains are low in gluten, which contribute to dough viscosity and extensibility (Dexter et al., 1997; Wang et al., 2005). Infected grains are high in free nitrogen and amino acids which results in intensive browning of the baked loaf surface. Further, Fusarium infection results into the withered and light test-weight kernels making marketing and processing of the grain difficult (Goswami and Kistler, 2004).

2.8 Epidemiology

The growth stage of the wheat plant is an important factor impacting infection, subsequent FHB symptom development and DON accumulation. Infection of wheat heads at the kernel watery ripe or early milk stage results in higher visually scabby kernel (VSK) and DON accumulation than infection occurring towards anthesis and late milk (Del Ponte et al., 2007). Wheat heads are however vulnerable to Fusarium infection until the dough stages (Anderson, 1948; Atanasoff, 1920) and cultivars differ in terms of their susceptibility at different growth stages (Schroeder and Christensen, 1963). Difference in the most susceptible stage, in terms of infection and DON accumulation, has also been shown in barley (Yoshida et al., 2007). Susceptibility of wheat at anthesis and thereafter has been often linked to the protrudence of anthers (Strange and Smith, 1971). Anthers are rich in choline and glycinebetaine which are reported to have stimulatory effect on fungal hyphal extension (Strange et al., 1974). Thus anthers may have role in stimulating fungal growth and providing a point of entrance into the spikelets. Other factors promoting susceptibility during anthesis are the temporary opening of florets allowing airborne inoculum and fungal hyphae to enter, and the expansion of crevices between palea and lemma due to enlargement of the caryopsis (Bushnell et al., 2003). Environmental factors, especially moisture and temperature are important for FHB development and DON accumulation. Each Fusarium species has its own environmental requirements for growth and development. Thus, even though several species can be detected in an FHB infested area, only a few species will predominate (Osborne and Stein, 2007). Generally, F. graminearum mycelia grow rapidly between 25ºC and 27ºC and are inhibited below 3ºC and above 35ºC (McInnes and Fogelmann, 1923). The optimum temperature for FHB infection and symptom development is 25ºC and FHB incidence increases rapidly when the temperature range from 20 to 30ºC (Andersen, 1948; Rossi et al. 2001). Similar reports of higher FHB symptoms evident at higher temperatures have been published by other researchers (Brennan et al., 2005). Environmental stresses influence FHB development and DON production which are often unrelated to total fungal biomass (Hope et al., 2005). Environmental factors triggering, regulating or influencing mycotoxin synthesis and accumulation in the infected host are not well understood (Mesterházy et al., 1999). Martins and Martins (2002) reported that more DON was produced on maize following incubation for 35 days at 28ºC compared to 22ºC. In the same experiment the production of DON decreased in incubation treatments longer than 35 days and DON was not detected in treatments at 37ºC.

The impact of moisture on FHB development and DON production and accumulation has been studied more than temperature. FHB epidemics appear to coincide with rainfall at and after flowering. McMullen et al. (1997) reported that in the epidemic year 1993, the month of July received the highest rainfall in wheat growing regions of South Dakota, North Dakota and Minnesota. In these regions in July wheat flowers and begins grain fill. Similarly, high rainfall was related to a FHB epidemic in 1980 in Canada (Sutton, 1982). Similar linkage between FHB and rainfall during anthesis or shortly thereafter are indicated by other studies (Abramson et al., 1987; Atanasoff, 1920; Rohácik and Hudec, 2005; Tuite et al., 1990). Hope et al. (2005) studied the relationship of water activity (aw) of the kernel with the Fusarium infection and DON accumulation. They indicated that below an aw of 0.90 (19-20% seed moisture), DON production by F. graminearum and F. culmorum was inhibited. However, Birzele et al. (2000) suggested that DON was produced in natural field grain at 17% grain moisture (aw of 0.8-0.85). Due to its prime role in FHB development and DON accumulation, several studies have studied moisture impact with extended periods of moisture either in the form of mist irrigation or bagging heads. Hart et al. (1984) studied the accumulation of DON in both greenhouse and field wheat in response to different moisture durations created by bagging heads with plastic bags after inoculation. The accumulation of DON increased with increased durations of bagging until 96 hours after infection in the greenhouse experiment. However, in field samples, DON accumulation decreased after 72 hours of bagging. Lacey et al (1999) also reported increased DON with increased moisture durations when they inoculated wheat heads with F. culmorum. However, they utilized continuous mist irrigation over 3-6 days after inoculation instead of plastic bags. Lemmens et al. (2004) found a differential reaction of wheat grain to DON accumulation with environment moisture. They tested two levels of mist-irrigation treatments (no irrigation and irrigation for 26 days after flowering) and observed a decrease in the DON level in some wheat lines receiving irrigation while observing an increase in others. Similarly, when Culler et al. (2007) applied mist- irrigation treatments and sampled multiple times after inoculation, they found less DON accumulation in the infected heads, especially in a susceptible variety, in the treatment mist- irrigated for longer durations when compared to the treatments receiving misting for shorter durations. Though the results were not significant for all sampling times, lower DON contamination was generally observed despite the increases in both disease levels and VSK. Cowger et al. (2009) reported variable results for FHB severity and DON to mist-irrigation treatments. In one year of the study FHB severity and DON increased with the duration of mist irrigation, while in next year both FHB severity and DON decreased in the longest misted treatment. Thus the impact of extended moisture period on FHB development and DON accumulation is not well understood. The impact of moisture appears to be compounded by temperature. Hart et al., (1984) reported the concentration of DON in the field lower compared to greenhouse studies, which the authors attributed to a possible spike in temperature inside the plastic bags used in the field. Several studies have indicated that with decreasing temperature, increased durations of moisture are required for FHB development (Pugh et al., 1933; Rossi et al., 2001). Thus the FHB incidence and severity, and final DON concentration in the infected heads is probably a result of complex interactions between the host, the pathogen and the environment.

Crop rotation and cropping patterns also are considered important factors influencing FHB development. It was reported that FHB incidence and DON concentrations were higher in winter wheat planted after corn than after soybeans in the province of Ontario in Canada (Teich and Hamilton, 1985). Corn and other cereals are also major hosts of F. graminearum. Thus wheat following corn or wheat in a rotation provides an opportunity for Fusarium to overwinter in residues and serve as inoculum for the next growing season (Wilcoxson et al., 1988). Several studies have shown evidence of increased FHB incidences and DON accumulation in wheat following wheat or wheat following corn (Holbert et al., 1919; Koehler et al., 1924; Windels and Kommedahl, 1984). The situation is further aggravated by the minimum or zero tillage practices, in which crop resides are left on the soil surface (Dill-Macky and Jones, 2000).

2.9 Genetics of FHB resistance

Resistance to FHB is conferred mainly by physiological and morphological components. Schroeder and Christensen (1963) reported two components of physiological resistance in wheat to FHB; type I resistance or the resistance against initial infection and type II or resistance against spread of infection within the spike. Type I resistance can be detected by spray inoculating heads and measuring the FHB incidence while type II resistance can be detected based on spread of infected spikelets upward and downward after single centrally located floret in a spike is inoculated. Wheat lines may possess either type I and/or type II (Schroeder and Christensen; 1963). Three other types of physiological resistances have been proposed; resistance to kernel infection (Mesterházy, 1995; Mesterházy et al., 1999), FHB tolerance (Mesterházy, 1995; Mesterházy et al., 1999) and resistance to toxin accumulation (Miller et al., 1985). However, a debate is still going on for codifying them in standardized list of resistance (Bushnell, 2002).

Variation in wheat genotypes for FHB resistance exists worldwide and the best resistances are generally found in three gene pools; spring wheats from Brazil, spring wheats from China and Japan, and winter wheats from Europe (Snijders, 1990). FHB resistant wheat cultivars include the Eastern European winter wheats ‘Arina’, ‘Praag-8’ and ‘Renan’. In spring wheat ‘Sumai-3’ and its derivatives from China, ‘Nobeoka Bozu’ and ‘Sin Chunaga’ and their derivatives from Japan, and ‘Frontana’ and ‘Encruzilhada’ from Brazil are reported to carry FHB resistance (Ruckenbauer et al., 2001; Snijders, 1990; Zhang et al., 2008). In addition, the winter wheat ‘Ernie’ and ‘Freedom’, and spring wheat ‘2375’ are considered the best US source of FHB resistance (Rudd et al., 2001). Zhang et al. (2008) analyzed 1035 spring wheat worldwide accessions from the United States Department of Agriculture (USDA) National Small Grains Collection, noting those with moderate level of disease in the field, European resistant lines generally showed higher level of resistance in terms of VSK and DON. Sumai 3 has been shown to have type II FHB resistance (Hartel et al., 2004; Kolb et al., 2001). Because of its high combining ability and yield, Sumai 3 and its derivatives have been widely utilized as sources of resistance by breeding programs in the US and worldwide (Bai and Shaner 1994; Ruckenbauer et al., 2001). One concern with the use of a few resistance sources is that this has narrowed down the genetic base, increasing the possibility of wheat becoming more vulnerable to changes in the pathogen population (Ruckenbauer et al., 2001). Thus searches for FHB resistant genes has begun in wild relative and progenitor to identify sources with comparable resistance levels to ‘Sumai 3’ (Hartel et al., 2004; Oliver et al., 2005; Oliver et al., 2007).

The genetics of FHB resistance is complex due to its polygenic nature. Studies have indicated that FHB resistance is controlled by two to six genes, complicating its study and breeding for resistant cultivars (Singh et al., 1995; Snijders, 1990; Van Ginkel et al., 1996). Due to its quantitative nature of inheritance, and that FHB resistance is highly influenced by environment it is difficult to estimate the number of gene or to identify quantitative trait loci (QTL) (Groth et al., 1999; Kolb et al., 2001). The first major QTL reported to confer FHB resistance was on chromosome 3B (Fhb1 syn. Qfhs.ndsu-3BS) detected in a population from the cross of resistant cultivar ‘Sumai3’ and the moderately susceptible cultivar ‘Stoa’ (Waldron et al., 1999). Other QTLs identified are on chromosome 2A (2AL), 4B (4BL) and 6B (6BS) (Waldron et al., 1999), 6A (6AS) and 3A (3AL) (Anderson et al., 2001). Among these QTLs, 3BS explained phenotypic variation from 15.4% (Waldron et al., 1999) to 60% (Buerstmayr et al., 2002).

Several QTLs have been reported to be linked with DON accumulation in FHB infected head. QTLs on chromosomes 2DS and 5AS were reported to be significantly associated with low DON independent of FHB severity (Somers et al., 2003). Similarly, Ma et al. (2006) reported a QTL on chromosome 5A, which explained 12.4% of DON variation independent of FHB severity in the recombinant inbred lines (RILs) from a cross of ‘Wangshuibai’ and ‘Annong 8455’. The same study also reported other QTLs on chromosomes 2A (minor effect on both FHB severity and DON) and 3B (major effect on FHB severity and minor effect on DON). Other QTLs reported are on 1AL and 2AS detected in double haploid population from a cross between ‘Arina’ and ‘NK93604’ (Semagn et al., 2007). QTL 1AL explained 27.9% variation in DON and was also associated with FHB severity. QTL 2AS explained 26.7% variation and was associated only with low DON. Recently Abate et al. (2008) reported three more QTLs associated with low DON and low Fusarium damaged kernel (FDK) in a RILs population from a cross between ‘Ernie’ and ‘MO 94-317’.

In addition to physiological mechanisms of FHB resistance, agronomic characteristics including short plant height and the presence of awns have also been linked to increased FHB incidence and severity (Buerstmayr et al., 2000; Mesterházy, 1995; Ransom, 2008). Higher spikelet density in heads has also been linked to a higher probability of FHB because of increased humidity in the head (Rudd et al., 2001). It is reported that, on average, FHB resistant wheat is associated with lower levels of DON than FHB susceptible wheat (Wilde and Miedaner, 2006). According to Arsenuik et al. (1999), the correlation is stronger (r = 0.74) for spring wheat than winter wheat (r = 0.54). In the same study, the deviation from linearity was higher for wheat than rye or triticale, when the relationship between FHB and DON was considered. In a greenhouse study by Bai et al. (2001) FHB ratings, in terms of the proportion of scabby kernels (PSS; r = 0.65) and area under disease progression curve (AUPDC; r = 0.75), were reported to be significantly and positively correlated with DON levels. Several other researchers have reported that resistant cultivars have lower DON levels than susceptible cultivars (Mesterházy et al., 2003; Miller et al., 1985; Wilde and Miedaner, 2006). Whether the reduced DON in cultivars expressing FHB resistance is due to the resistance of the host to initial infection, spread of the fungus in the spike (Schroeder and Christensen, 1963) or modes of resistance conferring either the prevention of DON synthesis or the degradation of DON (Miller et al., 1985; Miller and Arnison, 1986) has not been clearly established. To facilitate germplasm evaluation and selection, researchers utilize correlations between visual FHB severity assessments in the field and DON concentrations to predict DON in harvested grain (Arsenuik et al., 1999; Groth et al., 1999; Jones and Mirocha, 1999). Although correlations are frequently significant, correlation coefficients are often low and may vary greatly among locations and years. The reasons for this variability are not fully understood, but may be associated with the toxin production capacity of prevalent strains within the Fusarium population, the type and level of host plant resistance to FHB and/or environmental conditions prevailing between the time of initial infection and harvest.

2.10 FHB management

Because of the lack of FHB resistant wheat cultivars, chemical control has been widely utilized in the US over the past decade. The effectiveness of fungicides differs depending upon the environment and the cultivars upon which they are used (Hollingsworth et al., 2008). Several fungicides have been tested, among which tebuconazole, propiconazole, carbendazim, and benomyl have shown promising results (Mesterházy, 2003). However, fungicides results are not consistent. When Milus and Parsons (1994) tested several fungicides including tebuconazole and propiconazole, none of them reduced FHB severity and DON concentration or increased yield. However, Balaž et al. (2008) reported a significant reduction in FHB severity and an increase in yield. They treated wheat plants during flowering with several fungicides including epoxyconazole, thiophanate-methyl, carbendazim, flusilazole, trifloxystrobin, cyproconazole, tebuconazole, triadimenol, spiroxamine, procholraz, and propiconazole. Boyacioglu et al. (1992) reported reduction of DON by 80% with triadimefon, while propiconazole and thiobendazole were not effective in reducing DON. Several other researchers have indicated inconsistent fungicide results (Horsely et al., 2006; McMullen, 1994). Timing of application and application technology appear important for successful results when applying fungicides. Fungicides have also been tested for their effects on residue colonization by F. graminearum and have shown promising results to reduce residue colonization (Beare et al., 1993), although fungicides might also impact residue colonization by residue decomposing or other Fusarium -competitive microorganisms. Lack of accurate disease forecasting methods and the high cost of fungicide application limit the use of fungicides for FHB management (McMullen et al., 1997).

Tillage practices and residue management have been considered vital for the control of FHB epidemics as Fusarium is a residue borne pathogen. Dill-Macky and Jones (2000) showed that FHB incidence, severity and DON concentration was significantly higher in wheat following corn with no-till cultivation. Moldboard plowed plots had lower FHB incidence and severity than chisel plowed and no-tilled plots. Since moldboard plowing inverts the soil layer burying crop residues, the survival of Fusarium is reduced. Pereyra and Dill-Macky (2008) found that G. zeae can survive in several gramineae plant residues which subsequently act as inoculum sources. They showed that the survival of G. zeae decreased rapidly in wheat and barley residue compared to corn. Considering the fact that Fusarium survives in surface crop residues, especially gramineae crops, FHB may be managed by rotating with non-host crops and by proper burial of residues.

In the search for disease management strategies, biological control has emerged as one possibility. Several microorganism including bacteria (Bacillus spp., Kluyvera cryocrescens, Lysobactor spp., Paenibacillus fluorescens, Pantoea agglomerans, and Pseudomonas fluorescens), yeasts (Cryptococcus spp., Rhodotorula spp., and Sporobolomyces roseus) and filamentous fungi (Trichoderma harzianum and T. virens) have shown potential for the control of F. graminearum (Bacon and Hinton, 2007; Corio da luz et al., 2003; Jochum et al., 2006). However, problems encountered in biocontrol agents include their viability, delivery mechanisms, suitability of application with fungicides and inconsistent results (Yuen et al., 2007; Yuen, 2008).

It appears therefore that the development and use of resistant host is a most economical and environmentally safe strategy for disease management (Ruckenbauer et al., 2001). Efforts on FHB resistance breeding have been augmented worldwide, although progress in the development of FHB resistant cultivars has been slow. Several QTLs associated with FHB and DON resistances have been used to provide partial resistance to FHB. Efforts are ongoing for pyramiding multiple QTLs to a single cultivar (Shi et al., 2008). Similarly, several studies are focused on identifying transgenic wheat by incorporating plant defense antifungal proteins like thaumatine-like proteins (Chen et al., 1999; Mackintosh et al., 2007). Though the results of some of these studies have been promising in the greenhouse experiment, they have failed to prove significant in field environments (Anand et al., 2003). Wheat cultivars with partial resistance are available for commercial cultivation, but immune cultivars are lacking.

In conclusion it appears that FHB cannot be controlled by any single measure of fungicides, resistant cultivar or residue management strategies. An integrated approach utilizing several of the available mechanisms seems most likely to provide effective control of FHB (McMullen et al., 1997; Yuen and Schoneweis, 2007).

Chapter 3 Impact of Post-Inoculation Moisture on Fusarium Head Blight (FHB) Development and Trichothecene Accumulation in Spring Wheat

Fusarium head blight (FHB), primarily caused by Fusarium graminearum Schwabe, is an economically important disease as it results in yield and quality losses of infected grain and the accumulation of mycotoxins produced by the invading fungus. Environmental factors, host genetics and isolate aggressiveness impact FHB development and subsequently trichothecene production and accumulation. Though it is well established that moisture around the anthesis period promotes FHB development and trichothecene accumulation, the role of moisture, either in the form of rainfall or mist-irrigation during the period from anthesis to harvest has been largely overlooked. The objective of this study was to examine the influence of environmental factors, especially moisture, host resistance, and pathogen variation with respect to mycotoxin production capacity and aggressiveness, on infection, FHB disease development and mycotoxin production and accumulation in planta. The field experiments, conducted in 2006-2008, were a split-split- plot design with five replications. Main plots were the duration of mist-irrigation after inoculation [14, 21, 28 and 35 days after inoculation (DAI)]. Sub-plots were wheat cultivars. Three wheat cultivars used were Alsen (moderately resistant with the resistance derived from Sumai 3), 2375 (moderately susceptible) and Wheaton (susceptible). Sub-sub-plots were F. graminearum isolates (49-3, 81-2, B45A, B63A, and Butte86ADA-11) differing in terms of their aggressiveness and DON production capacity in addition to a mock-inoculated water control. Plots were inoculated twice, at anthesis and 3 days after the first inoculation with macroconidial inoculum (1×105 conidia ml-1 ). FHB severity was assessed 21 dai by counting the total and visually symptomatic spikelets in 20 arbitrarily selected heads per plot. Visually scabby kernels (VSK), deoxynivalenol (DON), 15-acetyldeoxynivalenol (15-ADON), 3-acetyldeoxynivalenol (3-ADON) and nivalenol (NIV) were determined on grain harvested at maturity. Additionally, in 2007 and 2008, whole heads (10 per plot) were sampled at 0, 7, 11, 14, 21, 28 and 41 dai. These heads were dried and the entire head ground and analyzed for DON, 15-ADON, 3-ADON and NIV. Severity, VSK and the DON concentration of mature grain, were significantly higher (P < 0.05), across all isolates, in the susceptible wheat cultivar Wheaton than in the other two cultivars examined. Although FHB severities were not significantly different in plots receiving different durations of mist-irrigation ,VSK were significantly lower (P < 0.05) in the treatments receiving the least amount of mist-irrigation (14 DAI) than for treatments receiving mist-irrigation for longer periods, suggesting that extended periods of moisture promote disease development. DON was however significantly lower (P < 0.05) in the 35 DAI misting treatment than those treatments receiving less water. In the whole head samples, DON and other trichothecene either declined with increased durations of mist-irrigation or remained low while water was being applied by the misting system. However, trichothecene accumulation was observed to increase after the cessation of mist-irrigation, with increases being most pronounced for the treatments with shorter mist-irrigation periods. The largest reduction in DON observed as a result of extended mist- irrigation periods was seen in the susceptible cultivar Wheaton. Our results suggest that longer durations of moisture after inoculation, either from mist-irrigation or rainfall, may increase the FHB severity and VSK, although DON and other trichothecene concentrations may be concomitantly reduced. Leaching may explain the reduction of DON observed in extended misting duration treatments.

3.1 Introduction

Fusarium head blight (FHB or scab), a destructive disease of wheat and other small grains in warm and humid cereal growing regions of the world, is primarily incited by F. graminearum Schwabe [teleomorph Gibberella zeae (Schwein) Petch] (Bai et al., 2001; Doohan et al., 2003; Schroeder and Christensen, 1963; Wilcoxson et al., 1992). The reemergence of FHB (Windels, 2000) was estimated to cause direct and secondary economic losses of $2.7 billion in wheat and barley in the northern Great Plains and central US from 1998 to 2000 (Nganje et al., 2002). Although FHB has been common, severe and well documented in the past, recent epidemics have resulted in increased concern, greater public interest and expanded research efforts.

Warm temperatures and extended periods of moisture on plant surfaces around anthesis favor the infection and colonization of wheat tissues by F. graminearum. In addition to yield losses, the quality of infected grain is compromised due to the production of a range of mycotoxins, including deoxynivalenol (DON), 15-acetyldeoxynivalenol (15-ADON), 3- acetyldeoxynivalenol (3-ADON), and nivalenol (NIV), by infecting Fusarium (Nasri et al., 2006; Pirgozliev et al., 2003). These mycotoxins are hazardous to humans and animals, thus making highly infected grain unfit for food or feed. Wheat from US production fields infected by Fusarium have routinely tested positive for most of these mycotoxins (Abramson et al., 1987; McMullen et al., 1997). Deoxynivalenol is considered the most important toxin produced by F. graminearum and is regulated in grain and wheat based food products by US federal agencies. 22

The US, Canada and several European countries have set less than 2 mg kg-1 as the acceptable limit of DON in wheat grain for human consumption (Bai et al., 2001). In addition Fusarium damaged grain tends to be withered with light test-weight kernels exhibiting weak dough properties and unsatisfactory baking quality, making marketing and processing of the grain difficult (Bechtel et al., 1985; Dexter et al., 1996; Dexter et al., 1997; Wang et al., 2005).

FHB resistant wheat is reported to be associated with lower levels of DON than FHB susceptible wheat (Wilde and Miedaner, 2006). According to Arsenuik et al. (1999), the correlation between FHB visual symptoms and DON was stronger (r = 0.74) for spring wheat than winter wheat (r = 0.54). Other researchers have also observed that resistant cultivars generally have lower DON levels than susceptible cultivars (Mesterházy et al., 2003; Miller et al., 1985; Wilde and Miedaner, 2006). Whether the reduced DON in cultivars expressing FHB resistance is due to the resistance of the host to initial infection, spread of the fungus in the spike (Schroeder and Christensen, 1963) or modes of resistance conferring either the prevention of DON synthesis or the degradation of DON (Miller et al., 1985; Miller and Arnison, 1986) has not been clearly established. To facilitate the evaluation and selection of resistant germplasm and to assess commercial crop quality, researchers frequently utilize correlations between visual FHB severity assessments in the field and DON concentrations to predict DON in harvested grain (Arsenuik et al., 1999; Groth et al., 1999; Jones and Mirocha, 1999). Although correlations are generally significant, correlation coefficients are frequently low and vary greatly among locations and years. The reasons for this variability are not fully understood, but may be associated with toxin production capacity of prevalent strains within the Fusarium population, the type and level of host plant resistance to FHB and/or the environmental conditions prevailing between the time of initial infection and harvest.

Environmental factors, primarily moisture, have been reported to have an important influence on toxin production and DON production is often unrelated to total fungal biomass (Hope et al., 2005). Moisture in the form of rainfall or relative humidity, during anthesis or shortly thereafter, has been linked to higher FHB incidences, higher FHB severities and DON accumulation (Abramson et al., 1987; Atanasoff, 1920; Rohácik and Hudec, 2005; Tuite et al., 1990). Epidemics are generally associated with the occurrence of rainfall during wheat anthesis (McMullen et al., 1997; Sutton, 1982). However, the specific environmental factors triggering, regulating or influencing mycotoxin synthesis and accumulation in the infected host are not well understood, (Mesterházy et al., 1999). The final toxin concentration in the kernel is likely the result of complex interactions between the host, the pathogen and the environments (Lemmens et al., 2004).

It has been reported that continuous mist irrigation over 3 days after inoculation with F. culmorum increased DON in harvested wheat (Lacey et al., 1999). Lemmens et al. (2004) reported differential DON accumulation in wheat grain when environmental moisture conditions differed. When Culler et al. (2007) applied mist-irrigation treatments and sampled multiple times after inoculation, he found less DON accumulated in the infected heads, especially in a susceptible variety, in the extended mist-irrigated treatment when compared to the standard mist- irrigation treatment. The DON results were, however, not significant at all sampling times despite observed increases in both disease levels and yield reduction in the extended mist-irrigation treatments. The results reported by Cowger et al., (2009) somewhat contradict those of Culler et al. (2007) and Lemmens et al. (2004). Cowger et al. (2009) observed increases in FHB severity, Fusarium damaged kernels and DON levels with increased durations of mist-irrigation from 0 to 30 days post-anthesis.

The role of DON in pathogen aggressiveness has not been well defined. While some studies have reported a positive correlation between aggressiveness and DON production by F. graminearum and F. culmorum (Gang et al., 1998; Hestbjerg et al., 2002; Mesterházy, 2002), others suggest no significant correlation or inconsistent correlation (Adam & Hart, 1989). However, there appears to be agreement that DON is not essential for pathogenesis (Dyer et al., 2005; Eudes et al., 2001; Proctor et al., 1995).

The pathogenic variability of F. graminearum has been well known since 1929 (Tu, 1929) and exists naturally in F. graminearum populations (Burgess et al., 1996). Variability in the capacity of F. graminearum with respect to aggressiveness and DON production has been reported both in vitro and in vivo (Tóth et al., 2005; Walker et al., 2001). An understanding of the impact of the diversity of F. graminearum on resistance expression and DON accumulation is useful for evaluating the risks of various FHB control strategies. Recent epidemics and emerging reports of changes in the types of mycotoxins produced by the Fusarium population (Gale et al., 2007; Jennings et al., 2004) have raised questions about the significance of changes in the genetic structure of the F. graminearum population at the regional and global scale. An assessment of the impact of the diversity and change in diversity of the pathogen population on FHB development is crucial if effective control practices are to be developed.

The objective of this study was to examine the influence of environmental factors, specifically moisture, host resistance, and pathogen variation with respect to mycotoxin production capacity and aggressiveness, on infection, FHB development and mycotoxin production and accumulation in planta.

3.2 Materials and methods

3.2.1 Inoculum production

Single isolates of F. graminearum representative of the range in aggressiveness and DON production capacity of the isolates available in the small grains pathology collection at University of Minnesota were used. Five different isolates (Appendix 1) were used; three collected from wheat fields (49-3, 81-2, and Butte86Ada-11) and two collected from barley fields (B63A, and B45A) in Minnesota. The isolates were maintained in soil and transferred to mung bean agar media (MBA: 40 g mung beans boiled for 23 minutes in 1000 ml of Millipore filtered water [screen size 0.22 μm; Milli Q Biocell, Millipore Corporation, France], filtered through two layers of gauze pads, adjusted to 1 L with Millipore filtered water, 15 g of Difco agar [Bectin, Dickinson and Company, Sparks, MD 21152] and autoclaved). Isolates in MBA were allowed to grow for 7 days under fluorescent and UV lights (12 h: 12 h light: dark cycle) at room temperature (22-24ºC). On the seventh day, ten milliliters of Millipore filtered water per plate was added in cultured isolate plates and rubbed with sterile L-shaped glass rod to loosen macroconidia. The ensuing spore suspension was filtered through a two layers of cheesecloth to reduce the number of mycelial fragments in the suspension and transferred into a sterile beaker and final volume was made equal to ca. 40 ml by adding Millipore filtered water. The spore suspension was used to inoculate 20 MBA plates per isolate (1.5 ml per plate), which were then incubated at room temperature for seven days as described earlier. On the seventh day, macroconidia were harvested by washing MBA plates with ca. 20 ml of deionized (DI) water per plate using a CO2 powered backpack sprayer fitted with flat-fan spray tip (TeeJet SS8003; Spraying Systems Co., Wheaton, IL) at an operating pressure of ca. 276 kPa. The spore suspension was filtered through one layer of cheesecloth to remove mycelia fragments. The spore concentration was determined using a hemacytometer and the inoculum concentration adjusted to 8×105 spores ml-1 inoculum was stored in 1L Nalgene® polyethylene bottles (Nalgene Nunc International Co., Rochester, NY) at - 80ºC until used for inoculation.

3.2.2 Field experiment design

Experiments were split-split-plots with five replications and established at the Experiment Station of University of Minnesota at Saint Paul, MN during the summers of 2006, 2007 and 2008. Main plots were mist irrigation treatments with four levels [mist irrigation from inoculation until 14, 21, 28, and 35 days after inoculation (DAI)], sub plots were the wheat genotypes and the sub-sub plots were the different isolates of F. graminearum. Cultivars within mist irrigation main plots and isolates within cultivar sub-plots were assigned randomly. Three wheat cultivars, varying in terms of resistance to FHB were used in the experiment; Alsen (Frohberg et al., 2006), previously identified as a source of FHB resistance and known to carry Fhb1 from the Chinese wheat Sumai 3; 2375 a moderately susceptible cultivar with a non-Asian source of resistance; and Wheaton (Busch et al., 1984) the susceptible check. Two row plots of the three wheat cultivars were planted in mid-April each year of the study. Each plot was 2 m long with the inter-row distance of 30 cm. In order to reduce the cross contamination of isolates, one row buffer plots of moderately resistant cultivar, Alsen, was planted between each experimental plot. Mist irrigation main plots were separated by six meters of an oat buffer. When wheat plants were at 3-4 leaf stage, the field was sprayed with the herbicide Bronate® (3,5-dibromo-4-hydroxybenzonitrile, 0.086 L a.i./ha; Bayer Crop Science, Research Triangle Park, NC) to control broadleaf weeds. Similarly, the insecticide, Di-syston 8® (disulfoton, 0.082 L a.i./ha; Bayer Crop Science, Research Triangle Park, NC), was sprayed to control aphids and other insects. Hand weeding throughout the growing season supplemented weed control. During the late booting stage, a micro sprinkle mist irrigation system (DAN 8000 series with rotating spinner, NETAFIM® Irrigation Inc., Altamonte Springs, FL) was assembled. Risers were at a height of 1 m. The mist-irrigation system was programmed, using a programmable timer, to run for 9 min every hour starting at 1700 and ending at 0700. The system ran for total of 14 misting periods each day or 126 minutes, and delivered 10.7 mm of water per day.

3.2.3 Inoculation

On the day of inoculation, stored inoculum was thawed and tested for germination by plating 0.5 ml of inoculum in potato dextrose agar (PDA) plates and counting germinated macroconidia after 8 hours. For inoculation, 1 L of inoculum (8×105 spores ml-1 ) was mixed with 7 L of water (making final concentration of 1×105 spores ml-1 ). Approximately 20 ml of Tween- 20 (polysorbate; Fisher Biotech, Fair Lawn, NJ) was added to the diluted inoculum as a wetting agent. All experimental plots were inoculated at anthesis (mid-June) and 3 days later. Inoculum was dispensed using a CO2-powered backpack sprayer operating at the pressure of ca. 276 kPa, fitted with flat-fan spray tip (TeeJet SS8003; Spraying Systems Co., Wheaton, IL), at the rate of 30 ml per meter of row. Mist irrigation was started immediately following inoculation for 10 min to prevent the rapid drying of inoculum. Mist irrigation treatments were imposed following the first inoculation.

Details

Pages
232
Year
2010
ISBN (Book)
9783640989539
File size
5.8 MB
Language
English
Catalog Number
v177203
Institution / College
University of Minnesota - Twin Cities
Grade
none
Tags
factors affecting fusarium head blight development trichothecene accumulation fusarium-infected wheat heads

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Title: Factors Affecting Fusarium Head Blight Development and Trichothecene Accumulation in Fusarium-infected Wheat Heads