Enhanced overall resistance to Fusarium seedling blight and Fusarium head blight in transgenic wheat by co-expression of anti-fungal peptides

Introduction of alien genes into wheat has been proposed as a strategy to breed cultivars with improved resistance to Fusarium seedling blight (FSB) and Fusarium head blight (FHB). In this study, we co-transformed different anti-fungal peptides (AFPs) into an elite wheat cultivar Yangmai11. We identified the genetically stable transgenic wheat lines carrying single or multiple genes by PCR, qRT-PCR and Southern blot analyses. Transgenic wheat lines 451 and 513 expressing two AFPs displayed a consistent, significantly improved overall resistance to FSB and FHB, whereas only FHB resistance was observed from other lines. Furthermore, crude proteins extracted from the lines 451 and 513 showed a clear inhibitory activity against F. graminearum in vitro. Taken together, it was essential to properly combine and express AFPs in transgenic wheat in order to obtain an improved overall resistance to Fusarium pathogens.     

Inverse effects of Arabidopsis NPR1 gene on fusarium seedling blight and fusarium head blight in transgenic wheat

In this study, the Arabidopsis thaliana NPR1 (non‐expressor of PR genes) gene was integrated into an elite wheat cultivar, and the response of the transgenic wheat expressing NPR1 to inoculation with Fusarium asiaticum was analysed. With seedling inoculation, the transgenic lines showed significantly increased fusarium seedling blight (FSB) susceptibility, whereas floret inoculation resulted in enhanced fusarium head blight (FHB) resistance. Quantitative real‐time PCR revealed that expression of two defence genes, PR3 and PR5, was associated with susceptible reactions to FSB and FHB, whereas the PR1 gene was activated in resistance responses. This inverse modulation by the constitutively expressed NPR1 gene suggests that NPR1 has a bifunctional role in regulating defence responses in plants. Therefore, it is unsuitable for improving overall resistance to FSB and FHB in wheat.     

The Brachypodium distachyon UGT Bradi5gUGT03300 confers type II fusarium head blight resistance in wheat

Fusarium head blight (FHB), caused by fungi belonging to the Fusarium genus, is a widespread disease of wheat (Triticum aestivum) and other small-grain cereal crops. The main causal agent of FHB, Fusarium graminearum, produces mycotoxins mainly belonging to type B trichothecenes, such as deoxynivalenol (DON), that can negatively affect humans, animals and plants. DON detoxification, mainly through glucosylation into DON-3-O-glucose, has been correlated with resistance to FHB. A UDP-glucosyltransferase from the model cereal species Brachypodium distachyon has been shown to confer resistance both to initial infection and to spike colonization (type I and type II resistances, respectively). Here, the functional characterization of transgenic wheat lines expressing the Bradi5g03300 UGT gene are described. The results show that, following inoculation with the fungal pathogen, these lines exhibit a high level of type II resistance and a strong reduction of mycotoxin content. In contrast, type I resistance was only weakly observed, although previously seen in B. distachyon, suggesting the involvement of additional host-specific characteristics in type I resistance. This study contributes to the understanding of the functional relationship between DON glucosylation and FHB resistance in wheat.     

Silicon amendment improves wheat defence against Fusarium graminearum and complements the control by fungicide of Fusarium head blight

A three-year field experiment with two wheat cultivars evaluated the effect of soil-applied silicon (Si), with and without fungicide spraying, on Fusarium head blight (FHB) control. Silicon treatment alone reduced FHB severity and the percentage of damaged wheat kernels, regardless of the cultivar. The best disease control was obtained for the cultivar with moderate disease resistance (MR), supplied with silicon and treated with fungicide during flowering. Silicon treatment alone promoted an increase in deoxynivalenol (DON) concentration in the disease-susceptible cultivar; however, in the MR cultivar, silicon amendment associated with fungicide treatment led to a reduction in DON concentration. Greenhouse experiments evaluated the effect of silicon combined with different timings of fungicide application on wheat defences against Fusarium graminearum. Plants supplied with silicon had a longer pathogen incubation period, lower FHB severity and lower DON concentration when compared to plants without silicon. In addition, silicon-supplied plants had higher soluble phenolic content and altered antioxidant enzyme activities (SOD, CAT, POX and PPO) that favoured early accumulation of hydrogen peroxide when compared to plants without silicon. Greater control of FHB and lower DON concentration in plants treated with silicon and fungicide before inoculation and up to 1 day after inoculation was associated with increased levels of defence-associated metabolites. Silicon contributed to the reduction of FHB and DON concentration in wheat, especially for the MR cultivar and, when combined with fungicide spraying, both MR and disease-susceptible cultivars had enhanced performances upon silicon amendment.     

Identification of fusarium head blight resistance related metabolites specific to doubled-haploid lines in barley

Fusarium head blight (FHB) and deoxynivalenol (DON) mycotoxin produced by Fusarium graminearum reduce barley yield and quality worldwide. Hundreds of quantitative trait loci (QTLs) have been identified in wheat and barley but their functions are largely unknown. Metabolic profiling was applied to better understand the mechanisms of resistance and to identify potential FHB resistance biomarker metabolites in barley. Four FHB resistant (H15-2, H148-3, H203-2 and H379-2) and one susceptible (H97-2), two-row, purple, doubled-haploid (DH) lines of barley were inoculated with either the pathogen or mock-solution. The disease severity quantified as the area under the disease progress curve (AUDPC) significantly varied between the resistant and susceptible genotypes, but not among the resistant genotypes. Neither the amount of DON nor the detoxified product, proportion of total DON, was significant among lines. The resistance related (RR, higher in abundance in resistant than in susceptible) metabolites varied in numbers and fold changes among the DH resistant lines. A total of 144 RR constitutive (RRC) and 167 RR induced (RRI) metabolites were selected, of which 39 and 37, respectively, were putatively identified. These RR metabolites mainly belonged to six chemical groups: phenylpropanoids, hydroxycinnamic acid amides, flavonoids, fatty acids, terpenoids, and alkaloids. The specific RR metabolites identified in each DH line, the possible mechanisms of resistance in each and their use as potential biomarkers are discussed.     

不同小麦品种(系)对赤霉病的抗性和麦穗组织中DON毒素积累分析

 为明确不同小麦品种(系)对赤霉病的抗性和麦穗组织中DON毒素积累水平,培育和利用抗赤霉病和DON毒素积累的品种提供资源和依据,本研究采用单小花滴注接种法对河南省的106个小麦品种(系)抗赤霉病性进行鉴定分析,并用ELISA测定了病穗组织中DON毒素水平。结果表明不同小麦品种(系)对赤霉病的抗性有显著差异,106个小麦品种(系)中未发现抗病和中抗材料,中感品种(系)有华育198、郑麦103和春丰0021等14个,占13.2%;感病的有曌式2010-06、百农898和中麦63等92个,占86.8%。不同小麦品种(系)籽粒、颖壳和穗轴中DON毒素积累水平有显著差异,籽粒中DON毒素水平在(0.70~287.63)mg/kg之间,其中郑03876、豫保1号和中麦63 的DON毒素水平在2 mg/kg 以下,为抗毒素材料;其他的103个品种DON毒素水平大于2 mg/kg;颖壳和穗轴中的DON毒素水平在(51.03~392.87)mg/kg之间,普遍比籽粒中DON毒素含量高。籽粒中DON毒素水平与小麦品种(系)的平均病害严重度间呈极显著正相关。     

Real-time PCR Quantification and Mycotoxin Production of Fusarium graminearum in Wheat Inoculated with Isolates Collected from Potato, Sugar Beet, and Wheat

ABSTRACT Fusarium graminearum causes Fusarium head blight (FHB) in small grains worldwide. Although primarily a pathogen of cereals, it also can infect noncereal crops such as potato and sugar beet in the United States. We used a real-time polymerase chain reaction (PCR) method based on intergenic sequences specific to the trichodiene synthase gene (Tri5) from F. graminearum. TaqMan probe and primers were designed and used to estimate DNA content of the pathogen (FgDNA) in the susceptible wheat cv. Grandin after inoculation with the 21 isolates of F. graminearum collected from potato, sugar beet, and wheat. The presence of nine mycotoxins was analyzed in the inoculated wheat heads by gas chromatography and mass spectrometry. All isolates contained the Tri5 gene and were virulent to cv. Grandin. Isolates of F. graminearum differed significantly in virulence (expressed as disease severity), FgDNA content, and mycotoxin accumulation. Potato isolates showed greater variability in producing different mycotoxins than sugar beet and wheat isolates. Correlation analysis showed a significant (P < 0.001) positive relationship between FgDNA content and FHB severity or deoxynivalenol (DON) production. Moreover, a significant (P < 0.001) positive correlation between FHB severity and DON content was observed. Our findings revealed that F. graminearum causing potato dry rot and sugar beet decay could be potential sources of inoculum for FHB epidemics in wheat. Real-time PCR assay provides sensitive and accurate quantification of F. graminearum in wheat and can be useful for monitoring the colonization of wheat grains by F. graminearum in controlled environments, and evaluating wheat germplasms for resistance to FHB.     

糖基转移酶基因TaUGT7分子表达特征及抗赤霉病作用分析

为研究小麦UDP-葡萄糖基转移酶7(UDP-glycosyltransferase 7,TaUGT7)的抗赤霉病功能,利用DNAMAN 6.0软件对Ta UGT7及其同源蛋白进行序列比对,应用实时荧光定量PCR(quantitative real-time PCR,qRT-PCR)技术分析经赤霉菌Fusarium graminearum和脱氧雪腐镰刀菌烯醇（deoxynivalenol,DON）处理后的苏麦3号小穗中TaUGT7基因的表达特征,利用基因枪在洋葱表皮细胞瞬时表达TaUGT7-eGFP进行亚细胞定位,采用农杆菌介导法在小麦品种Fielder中过量表达TaUGT7基因并进行赤霉病抗性鉴定。结果表明,TaUGT7在氨基酸序列上与已知赤霉病抗性相关UGT相似性较低;TaUGT7在赤霉菌接种24 h后开始被诱导表达,在DON处理2 h后逐步被诱导表达;Ta UGT7蛋白亚细胞定位于细胞膜和细胞核中;qRT-PCR检测发现,TaUGT7在8株独立的过表达转基因株系中均有不同程度的上调表达;与野生型对照相比,过表达株系TaUGT7-395和TaUGT7-457中的平均病小穗率显著下降。...     

Assessment of infection in wheat by <Emphasis Type="Italic">Fusarium</Emphasis> protein equivalent levels

Determination of the Fusarium protein equivalent (FPE) levels in kernels for better characterisation of genotypes showing Fusarium head blight (FHB) resistance, and better detection of susceptibility to kernel infection among genotypes with slight symptom expression was carried out. Twelve wheat cultivars and eight hexaploid winter wheat lines derived from a cross of Triticum aestivum with related species T. macha, T. polonicum, and T. dicoccoides were evaluated for levels of spike and kernel infection, the content of the mycotoxin deoxynivalenol (DON) and FPE in kernels after artificial inoculation with the fungus Fusarium culmorum in the field in 2006–2007. The ELISA immunochemical method was employed for the quantitative analyses of DON and FPE. Three wheat lines had a significantly low infection of spikes and kernels compared to cvs Sumai 3 and Nobeoka Bozu, indicating the presence of specific resistance mechanisms to FHB. The significantly low AUDPC (area under the disease progress curve) and the high level of FPE and DON content in kernels indicated a lack of resistance in one wheat line (crossed with T. polonicum). The results showed highly significant correlations (P < 0.01) between FPE and DON content and between FPE and AUDPC. In addition, correlations between FPE and reductions in yield components were also highly significant. Quantification of Fusarium spp. in wheat kernels can be helpful for evaluating wheat genotypes for their levels of resistance to FHB.     

小麦不同生育期施用丙硫菌唑及施用次数对小麦赤霉病及籽粒DON毒素的控制效果

为探明在小麦不同生育期施用新型杀菌剂丙硫菌唑及施用次数对小麦赤霉病及籽粒DON毒素总量[包括脱氧雪腐镰刀菌烯醇(DON)?3-乙酰脱氧雪腐镰刀菌烯醇(3-ADON)?15-乙酰脱氧雪腐镰刀菌烯醇(15-ADON)]的控制效果, 2020年-2021年通过田间自然发病和人工接种发病试验的方法, 研究了小麦不同生育期施用丙硫菌唑及施用次数对小麦赤霉病的防效, 并通过液相色谱-串联质谱法测定了不同处理小麦籽粒中对DON毒素总含量?结果表明, 30%丙硫菌唑可分散油悬浮剂(OD)防治2次对小麦赤霉病的防效和对DON毒素的控制效果均显著高于防治1次; 防治2次时, 其首次最佳防治时期为小麦齐穗期至扬花20%, 防效为90.25%~95.13%, 毒素控制效果为77.35%~79.97%?30%丙硫菌唑OD作为防控小麦赤霉病的新型药剂具有良好的应用前景, 本研究为该药剂推广应用于小麦赤霉病及籽粒DON毒素的防控提供了科学依据?     

Resistance in winter wheat lines to deoxynivalenol applied into florets at flowering stage and tolerance to phytotoxic effects on yield

In Europe and North America, deoxynivalenol (DON) is the most prevalent mycotoxin associated with wheat head blight caused by Fusarium graminearum and Fusarium culmorum. Because DON is toxic to plants and enhances the ability of the pathogen to spread within a spike, wheat lines with resistance to DON should be more resistant to head blight. Resistance to DON has been associated with resistance gene Fhb1 that confers resistance to spread within a spike. The objectives of this study were to determine if wheat lines resistant to head blight were also resistant to DON, if genes other than Fhb1 confer resistance to DON, and to identify lines able to fill grain in the presence of DON. Susceptible controls and diverse North American and European winter wheat lines with resistance to head blight were screened for molecular markers linked to known head blight resistance genes, and evaluated in a greenhouse for resistance to DON and relative yield after application of DON to spikes at flowering. Fhb1 appeared to have the unique ability to confer resistance to DON, as measured by the number of DON‐bleached primary florets. However, this resistance did not protect plants from the phytotoxic effects of DON on kernel formation as measured by the relative yield of treated spikes. Furthermore, measuring the relative yield loss following DON application may be useful for identifying lines with tolerance to head blight.     

Evaluation of type I fusarium head blight resistance of wheat using non-deoxynivalenol-producing fungi

A series of experiments was conducted to determine whether type I resistance (resistance to initial infection) to fusarium head blight (FHB) in wheat could be assessed using fungal species/isolates that do not produce deoxynivalenol (DON), a mycotoxin critical to the spread of Fusarium graminearum in the wheat spike. It was shown that, while the non-toxin-producing species Microdochium nivale and M. majus could infect following spray inoculation of wheat spikes, they were unable to spread within the spike following point inoculation. However, although these species might reveal type I resistance, they are not highly pathogenic towards wheat. A nivalenol (NIV)-producing isolate of F. graminearum caused high levels of disease following spray inoculation, but spread only very slowly within the spike and rarely induced bleaching above the point of inoculation. It is proposed that spray inoculation with an appropriate, aggressive, non-DON-producing FHB pathogen may be used to characterize type I resistance to complement point inoculation with a DON-producing isolate to assess type II resistance (resistance to spread within the spike).     

The Effect of Inoculation Treatment and Long-term Application of Moisture on Fusarium Head Blight Symptoms and Deoxynivalenol Contamination in Wheat Grains

Fusarium head blight (FHB) is an important disease of wheat, which can result in the contamination of grains with mycotoxins such as deoxynivalenol (DON). Artificial inoculation of flowering ears with conidial suspensions is widely used to study FHB diseases. Our goal was to compare four inoculation treatments in which a conidial suspension was sprayed on flowering ears and to study the effect of the application of moisture during kernel setting and filling with a mist-irrigation system. Ten wheat genotypes were inoculated with a DON-producing Fusarium culmorum strain. Inoculation treatments varied in time of application of the inoculum (morning or evening) and in the method of controlling humidity during inoculation (bagging or mist irrigation). A wet season was simulated with a mist-irrigation system, keeping the crop canopy wet for at least 26 days after flowering. The severity of FHB symptoms (area under disease progress curve (AUDPC)), yield loss and DON contamination in the grains were determined. AUDPC data obtained with the different inoculation treatments were highly correlated (r=0.85–0.95). Mist irrigation after inoculation resulted in a higher mean disease severity, but in a overall lower toxin contamination as compared to the non-irrigated treatments. Genotypic differences in DON accumulation were present: for one wheat line toxin contamination significantly increased when irrigated, while two genotypes accumulated significantly less toxin. The closest relationships (r=0.73–0.89) between the visual symptoms and the DON content were obtained under moderate mean infection pressure. This relation between visual symptoms and the DON content deteriorated at higher infection levels.     

A comparison of aggressiveness and deoxynivalenol production between Canadian <Emphasis Type="Italic">Fusarium graminearum</Emphasis> isolates with 3-acetyl and 15-acetyldeoxynivalenol chemotypes in field-grown spring wheat

Twenty four isolates of Fusarium graminearum, half of which were 3-acetyldeoxynivalenol (3-ADON) and half 15-acetyldeoxynivalenol (15-ADON) chemotypes, were tested for their ability to produce deoxynivalenol and to cause Fusarium head blight (FHB) in spring wheat cultivars. The objectives of this study were to determine (1) whether 3-ADON isolates differ in aggressiveness, as measured by the FHB index, and DON production from 15-ADON isolates under field conditions, and (2) whether the performance of resistant host cultivars was stable across isolates. Field tests of all isolates were conducted with three replicates at each of two locations in Canada and Germany in 2008 with three host genotypes differing in FHB resistance level. The resistant host genotype showed resistance regardless of the chemotype or location. The differences between mean FHB indices of 3-ADON and 15-ADON isolates were not significant for any wheat genotype. In contrast, average DON production by the 3-ADON isolates (10.44 mg kg⁻¹) was significantly (P < 0.05) higher than for the 15-ADON isolates (6.95 mg kg⁻¹) at three of the four locations where moderately resistant lines were tested, and at both locations where susceptible lines were evaluated. These results indicate that 3-ADON isolates could pose a greater risk to food safety. However, as the mean aggressiveness and DON production of 3-ADON and 15-ADON chemotypes was similar on highly resistant lines, breeding and use of highly resistant lines is still the most effective measure of reducing the risks associated with DON in wheat.     

Mapping of quantitative trait Loci for fusarium head blight resistance in barley

ABSTRACT Fusarium head blight (FHB) is a devastating disease that causes significant reductions in yield and quality in wheat and barley. Barley grains infected with deoxynivalenol (DON), a vomitoxin produced by Fusarium graminearum, are rejected for malting and brewing. Among six-rowed barley cultivars tested thus far, only cv. Chevron exhibited resistance. This study was conducted to map genes and to identify DNA markers for marker-assisted breeding for FHB resistance in cv. Chevron with restriction fragment length polymorphism (RFLP) markers. A doubled haploid (DH) population was created from a cross between cv. Chevron and susceptible cv. Stander. Seven field experiments were conducted in four different locations in 2 years. A RFLP map containing 211 loci and covering over 1,000 centimorgans (cM) of the genome was used to map quantitative trait loci (QTL) associated with relatively low FHB severity and DON concentration. Morphological traits differing between the parents were also measured: heading date, plant height, spike angle, number of nodes per cm of rachis in the spike, and kernel plumpness. Many of the QTL for FHB and DON coincided with QTLs for these morphological traits. The "fix-QTL" algorithm in Mapmaker QTL was used to remove the part of the variance for FHB resistance that may be explained by heading date or plant height. Results from this study suggest that QTLs with major effects for FHB resistance probably do not exist in cv. Chevron. Three QTL intervals, Xcmwg706-Xbcd441 on chromosome 1H, Xbcd307b-Xcdo684b on chromosome 2H, and Xcdo959b-Xabg472 on chromosome 4H, that are not associated with late heading or height may be useful for marker-assisted selection.     

Barley traits associated with resistance to fusarium head blight and deoxynivalenol accumulation

ABSTRACT Fusarium head blight (FHB) or scab is a destructive disease of barley in many countries. A better understanding of the interrelationships between plant traits and FHB resistance should help in the development of effective and efficient breeding strategies for FHB-resistant cultivars. Recent mapping studies indicate that many of the quantitative trait loci (QTL) for FHB resistance coincide with the QTL for plant height, heading date, and spike characteristics. Therefore, a study was conducted to investigate the relationship of morphological and physiological traits to FHB infection and deoxynivalenol (DON) accumulation in a barley doubled-haploid (DH) population derived from a Léger x CI9831 cross. Approximately 190 DH lines were grown at Ottawa (Ontario) for 2 years, Charlottetown (Prince Edward Island) for 1 year, and Hangzhou (Zhejiang) for 2 years. The field plots were inoculated with Fusarium graminearum at each location. FHB incidence was positively correlated with DON content. Resistance to FHB was associated with two-row spike, purple lemma, long glume awn, tall stature, and resistance to lodging, but it was not associated with long rachilla hairs, rough lemma awn, or heading date. Two-row spike was associated with tall stature and resistance to lodging. These associations as well as its spike characteristics helped reduce FHB infection and DON accumulation in two-row lines compared with six-row lines. The association between long glume awn and FHB resistance could be due to genetic linkages. Therefore, trait associations should be taken into consideration when breeding for FHB resistance and interpreting data from FHB experiments.     

Within-field variation of Fusarium graminearum isolates for aggressiveness and deoxynivalenol production in wheat head blight

Fusarium head blight (FHB), caused by Fusarium graminearum sensu stricto (s.s.), causes tremendous annual yield losses in wheat worldwide. Variation of aggressiveness of isolates from individual field populations in terms of FHB infection and deoxynivalenol (DON) concentration in the host are important population parameters reflecting parasitic ability. Our main objective was to estimate the variation of both traits within three populations of F. graminearum s.s., each consisting of 30 single-spore isolates collected from small wheat fields in Germany, and to compare it with 11 isolates of a collection (F. graminearum collection) from four countries. The same isolates were characterized using 19 single-sequence repeat markers. All isolates were spray inoculated on a moderately resistant spring wheat cultivar at two field locations over 2 years (i.e., in four environments). The genotypic proportion of phenotypic variance (σ(2)(G)) within populations was significant (P < 0.01) for both traits, and the σ(2)(G) × environment interaction was even more important for mean FHB severity. Ranges in mean FHB severity and DON concentration in the host were only slightly smaller for the field populations than for the F. graminearum collection. Both traits were significantly (P < 0.05) correlated within and across populations. A further partitioning of σ(2)(G) revealed 72% of σ(2)(G) within and 28% of σ(2)(G) across populations for both traits. Molecular variance of the three populations was similarly distributed (73.6% within versus 26.4% between populations). In view of this high within-field variation for traits of parasitic ability and selection, neutral molecular markers, multiple resistance genes of different origin should be employed in wheat breeding programs to obtain a long-term stable FHB resistance.     

Host genetic effect on deoxynivalenol accumulation in fusarium head blight of barley

ABSTRACT One of the major concerns with Fusarium head blight (FHB) of barley is the potential health risks to livestock and humans through the accumulation of the mycotoxin deoxynivalenol (DON) in infected grain. To define the role of the host in DON accumulation during the early stages of disease development, we conducted a series of greenhouse experiments. We inoculated single spikelets of greenhouse-grown plants with Fusarium graminearum, moved the plants to a dew chamber, and harvested the inoculated spikelets after 72 h for DON analysis. We conducted a quantitative trait locus (QTL) analysis using a genetic mapping population, constructed with the parents Stander and Frederickson, that segregated for DON accumulation after single-spikelet inoculation in two experiments. A single QTL on chromosome 3 explained 18 and 35% of the phenotypic variation in the two experiments. To validate this QTL for DON accumulation, we used a DNA marker to select near-isogenic lines from a family from the mapping population that was segregating at this QTL. Disease symptom development was similar between the nearisogenic lines; however, the mean DON concentration of the lines homozygous for the allele from the high DON parent was 2.5-fold more than the lines homozygous for the alternate allele. A time course experiment showed that this effect on toxin accumulation was observed at 10 days post inoculation. The near-isogenic lines developed in this study should prove useful for further exploration of the role of DON in FHB.