Transgenic wheat expressing a barley class II chitinase gene has enhanced resistance against Fusarium graminearum

Fusarium head blight (FHB; scab), primarily caused by Fusarium graminearum, is a devastating disease of wheat worldwide. FHB causes yield reductions and contamination of grains with trichothecene mycotoxins such as deoxynivalenol (DON). The genetic variation in existing wheat germplasm pools for FHB resistance is low and may not provide sufficient resistance to develop cultivars through traditional breeding approaches. Thus, genetic engineering provides an additional approach to enhance FHB resistance. The objectives of this study were to develop transgenic wheat expressing a barley class II chitinase and to test the transgenic lines against F. graminearum infection under greenhouse and field conditions. A barley class II chitinase gene was introduced into the spring wheat cultivar, Bobwhite, by biolistic bombardment. Seven transgenic lines were identified that expressed the chitinase transgene and exhibited enhanced Type II resistance in the greenhouse evaluations. These seven transgenic lines were tested under field conditions for percentage FHB severity, percentage visually scabby kernels (VSK), and DON accumulation. Two lines (C8 and C17) that exhibited high chitinase protein levels also showed reduced FHB severity and VSK compared to Bobwhite. One of the lines (C8) also exhibited reduced DON concentration compared with Bobwhite. These results showed that transgenic wheat expressing a barley class II chitinase exhibited enhanced resistance against F. graminearum in greenhouse and field conditions.     

Deoxynivalenol-nonproducing Fusarium graminearum Causes Initial Infection,but does not Cause DiseaseSpread in Wheat Spikes

Fusarium graminearum is a major pathogen that causes fusarium head blight (FHB) in wheat and produces deoxynivalenol (DON) in infected grain. In previous studies, the trichodiene synthase gene (Tri5) in the fungal strain GZ3639 was disrupted to produce the DON-nonproducing strain GZT40.In this report, the virulence of strains GZ3639 and GZT40 was tested on wheat cultivars with various resistance levels by using methods of spray inoculation and injection inoculation with fungal conidia. Under field and greenhouse conditions, strain GZ3639 produced significantly more disease symptoms and reduced more yield than strain GZT40 in all wheat cultivars tested. Conidia of strain GZT40 germinated and infected inoculated spikelets, but disease symptoms were limited to inoculated spikelets without spread to uninoculated spikelets. When strain GZT40 was inoculated using the spray method, multiple initial infection sites in a spike resulted in higher levels of disease symptoms than in spikes inoculated by a single injection. Greenhouse tests confirmed that strain GZT40 did not produce DON in the infected kernels following either inoculation method. The results confirm that DON production plays a significant role in the spread of FHB within a spike, and are the first report that DON production is not necessary for initial infection by the fungus. This revised version was published online in June 2006 with corrections to the Cover Date.     

Transgenic Arabidopsis thaliana expressing a barley UDP-glucosyltransferase exhibit resistance to the mycotoxin deoxynivalenol

Fusarium head blight (FHB), caused by Fusarium graminearum, is a devastating disease of small grain cereal crops. FHB causes yield reductions and contamination of grain with trichothecene mycotoxins such as deoxynivalenol (DON). DON inhibits protein synthesis in eukaryotic cells and acts as a virulence factor during fungal pathogenesis, therefore resistance to DON is considered an important component of resistance against FHB. One mechanism of resistance to DON is conversion of DON to DON-3-O-glucoside (D3G). Previous studies showed that expression of the UDP-glucosyltransferase genes HvUGT13248 from barley and AtUGt73C5 (DOGT1) from Arabidopsis thaliana conferred DON resistance to yeast. Over-expression of AtUGt73C5 in Arabidopsis led to increased DON resistance of seedlings but also to dwarfing of transgenic plants due to the formation of brassinosteroid-glucosides. The objectives of this study were to develop transgenic Arabidopsis expressing HvUGT13248, to test for phenotypic changes in growth habit, and the response to DON. Transgenic lines that constitutively expressed the epitope-tagged HvUGT13248 protein exhibited increased resistance to DON in a seed germination assay and converted DON to D3G to a higher extent than the untransformed wild-type. By contrast to the over-expression of DOGT1 in Arabidopsis, which conjugated the brassinosteriod castasterone with a glucoside group resulting in a dwarf phenotype, expression of the barley HvUGT13248 gene did not lead to drastic morphological changes. Consistent with this observation, no castasterone-glucoside formation was detectable in yeast expressing the barley HvUGT13248 gene. This barley UGT is therefore a promising candidate for transgenic approaches aiming to increase DON and Fusarium resistance of crop plants without undesired collateral effects.     

Response to Fusarium culmorum inoculation in barley

In field tests replicated in 2004 and 2005, 32 cultivars of spring barley were assessed for resistance to Fusarium head blight (FHB) by single floret inoculation and spray inoculation with Fusarium culmorum (W. G. Smith) Sacc. It was found that the weather conditions in individual years affect to a large extent the progression of FHB and production of mycotoxin deoxynivalenol (DON). At the same time, in both years the cultivars reacted to F. culmorum infection similarly with respect to areas under disease progress curve (AUDPC) values and content of mycotoxin DON. Spraying inoculation led to stronger infection. The biggest differences in AUDPC values were observed between the cultivars Brise and Celinka, and weak reaction was found in the cultivars Kompakt and Madonna. The cultivars Kompakt and Tolar were most resistant towards FHB. In both monitored years the variety Ludan contained the lowest amounts of mycotoxin DON. Cultivars with high infection and low DON content (r = 0.78) showed weak positive relationship between resistance to FBH and accumulation of DON (concentration 70–200 mg/kg). This is the first information on FHB and in vivo concentrations of DON in certificated barley cultivars in Slovakia.     

Identification of metabolites related to mechanisms of resistance in barley against Fusarium graminearum, based on mass spectrometry

Fusarium head blight (FHB) is an economically important disease of the family Triticeae, as, apart from yield reduction it also causes quality deterioration by producing mycotoxins. Host resistance is the most promising way to control the disease. Metabolic profiling was applied to identify resistance related (RR) metabolites against Fusarium graminearum in five FHB-resistant genotypes ('Chevron', 'H5277-44', 'H5277-164', 'M92-513' and 'M122') relative to one FHB-susceptible genotype ('Stander'). The disease severity was assessed in greenhouse to group the genotypes based on FHB-resistance. The disease was quantified as the proportion of diseased spikelets (PSD) and the area under the disease progress curve (AUDPC). Spikelets were collected at 72?h post inoculation. Metabolites were extracted into an aqueous solution of methanol and analyzed using a LC-hybrid-MS system. Metabolite abundances were subjected to a resistant versus susceptible pair-wise analysis, using a t test. Resistance related (RR) metabolites, both constitutive (RRC) and induced (RRI), were identified amongst metabolites whose levels were significantly higher in resistant genotype than in susceptible. Among 1,430 RR metabolites, 115 were putatively identified. These RR metabolites belonged to different chemical groups: fatty acids: linolenic acid; phenylpropanoids: p-coumaric, sinapic acid; flavonoids: naringenin, kaempferol glucoside, catechol glucoside. In addition, resistance indicator metabolites, such as deoxynivalenol (DON) and DON-3-O-glucoside (D3G) were also detected. The amount of total DON synthesized converted to D3G (PDC) was the greatest in resistant genotype 'Chevron' (PDC?=?0.76). The role of the resistance-related and resistance-indicator metabolites on plant defense, and their use as potential biomarkers to screen barley genotypes for FHB resistance is discussed.     

Comparison of the efficacy of chitosan with that of a fluorescent pseudomonad for the control of Fusarium head blight disease of cereals and associated mycotoxin contamination of grain

Fusarium culmorum can cause Fusarium head blight (FHB) disease of cereals, resulting in yield loss and contamination of grain with the trichothecene mycotoxin, deoxynivalenol (DON). In this study, we compared the efficacy of a biological control agent (Pseudomonas fluorescens strain MKB 158) with the biochemical chitosan (the deacetylated derivative of chitin) in controlling FHB disease of wheat and barley. Both agents were equally effective in reducing DON contamination of grain caused by F. culmorum. Under both glasshouse and field conditions, treatment with commercially available crabshell-derived chitosan reduced the severity of FHB symptom development on wheat and barley by ?74% (P ? 0.050). While treatment with P. fluorescens reduced the severity of FHB symptom development on these cereals by ?48% (P ? 0.050). Chitosan and P. fluorescens respectively prevented ?58 and ?35% of the FHB-associated reductions in 1000-grain weight in wheat and barley (P ? 0.050). Both agents significantly reduced the DON content of wheat and barley under both glasshouse and field conditions (P ? 0.050) and were equally efficacious in doing so (?74 and ?79% reductions due to chitosan and P. fluorescens, respectively). Crude chitin extracts from crabshells and crude chitosan-based formulations prepared from crabshells and eggshells were also tested under field conditions, but were not as effective as the commercial crabshell-derived preparation in controlling FHB disease. This is the first report on the use of chitosan for the control of Fusarium head blight disease and DON contamination of grain.     

Reduced susceptibility to Fusarium head blight in Brachypodium distachyon through priming with the Fusarium mycotoxin deoxynivalenol

The fungal cereal pathogen Fusarium graminearum produces deoxynivalenol (DON) during infection. The mycotoxin DON is associated with Fusarium head blight (FHB), a disease that can cause vast grain losses. Whilst investigating the suitability of Brachypodium distachyon as a model for spreading resistance to F. graminearum, we unexpectedly discovered that DON pretreatment of spikelets could reduce susceptibility to FHB in this model grass. We started to analyse the cell wall changes in spikelets after infection with F. graminearum wild‐type and defined mutants: the DON‐deficient Δtri5 mutant and the DON‐producing lipase disruption mutant Δfgl1, both infecting only directly inoculated florets, and the mitogen‐activated protein (MAP) kinase disruption mutant Δgpmk1, with strongly decreased virulence but intact DON production. At 14 days post‐inoculation, the glucose amounts in the non‐cellulosic cell wall fraction were only increased in spikelets infected with the DON‐producing strains wild‐type, Δfgl1 and Δgpmk1. Hence, we tested for DON‐induced cell wall changes in B. distachyon, which were most prominent at DON concentrations ranging from 1 to 100 ppb. To test the involvement of DON in defence priming, we pretreated spikelets with DON at a concentration of 1 ppm prior to F. graminearum wild‐type infection, which significantly reduced FHB disease symptoms. The analysis of cell wall composition and plant defence‐related gene expression after DON pretreatment and fungal infection suggested that DON‐induced priming of the spikelet tissue contributed to the reduced susceptibility to FHB.     

Quantitative trait loci associated with resistance to Fusarium head blight and kernel discoloration in barley

Resistance to Fusarium head blight (FHB), deoxynivalenol (DON) accumulation, and kernel discoloration (KD) in barley are difficult traits to introgress into elite varieties because current screening methods are laborious and disease levels are strongly influenced by environment. To improve breeding strategies directed toward enhancing these traits, we identified genomic regions containing quantitative trait loci (QTLs) associated with resistance to FHB, DON accumulation, and KD in a breeding population of F_4:7 lines using restriction fragment length polymorphic (RFLP) markers. We evaluated 101 F_4:7 lines, derived from a cross between the cultivar Chevron and an elite breeding line, M69, for each of the traits in three or four environments. We used 94 previously mapped RFLP markers to create a linkage map. Using composite interval mapping, we identified 10, 11, and 4 QTLs associated with resistance to FHB, DON accumulation, and KD, respectively. Markers flanking these QTLs should be useful for introgressing resistance to FHB, DON accumulation, and KD into elite barley cultivars. Received: 8 November 1998 / Accepted: 8 January 1999     

Quantification of trichothecene-producing Fusarium species in harvested grain by competitive PCR to determine efficacies of fungicides against Fusarium head blight of winter wheat

We developed a PCR-based assay to quantify trichothecene-producing Fusarium based on primers derived from the trichodiene synthase gene (Tri5). The primers were tested against a range of fusarium head blight (FHB) (also known as scab) pathogens and found to amplify specifically a 260-bp product from 25 isolates belonging to six trichothecene-producing Fusarium species. Amounts of the trichothecene-producing Fusarium and the trichothecene mycotoxin deoxynivalenol (DON) in harvested grain from a field trial designed to test the efficacies of the fungicides metconazole, azoxystrobin, and tebuconazole to control FHB were quantified. No correlation was found between FHB severity and DON in harvested grain, but a good correlation existed between the amount of trichothecene-producing Fusarium and DON present within grain. Azoxystrobin did not affect levels of trichothecene-producing Fusarium compared with those of untreated controls. Metconazole and tebuconazole significantly reduced the amount of trichothecene-producing Fusarium in harvested grain. We hypothesize that the fungicides affected the relationship between FHB severity and the amount of DON in harvested grain by altering the proportion of trichothecene-producing Fusarium within the FHB disease complex and not by altering the rate of DON production. The Tri5 quantitative PCR assay will aid research directed towards reducing amounts of trichothecene mycotoxins in food and animal feed.     

Sanitary factors and mycotoxin contamination in the argentinian wheat crop 1993/94

In Argentina, due to climatic conditions, Fusarium head blight (FHB) caused by Fusarium graminearum, affected the 1993/94 wheat crop. To evaluate the severity of this disease, samples of wheat where gathered from four zones of the wheat area. Sanitary conditions and mycotoxin contamination were determined. One zone (IIN) was intensely affected by FHB with 90% of samples in grade III (bad quality). No samples were grade I (good quality). The other zones were less affected falling into grade I or II (moderate quality). In all samples tested F. graminearum was the most prevalent species singly or in combination with others. Zone II N, with a DON mean level of I1.26 ppm, did not fulfil aceptability limits, whereas zones IIS, III and IV with overall means of 2.12, 1.57 and 1.0 ppm, respectively, did. Statistical analysis showed a close relation between percentage FHB and DON contamination (r:-0.71, p<0.01) in infected samples.     

Molecular mapping of novel genes controlling Fusarium head blight resistance and deoxynivalenol accumulation in spring wheat.

Fusarium head blight of wheat is an extremely damaging disease, causing severe losses in seed yield and quality. The objective of the current study was to examine and characterize alternate sources of resistance to Fusarium head blight (FHB). Ninety-one F1-derived doubled haploid lines from the cross Triticum aestivum 'Wuhan-1' x Triticum aestivum 'Maringa' were examined for disease reaction to Fusarium graminearum by single-floret injection in replicated greenhouse trials and by spray inoculation in replicated field trials. Field and greenhouse experiments were also used to collect agronomic and spike morphology characteristics. Seed samples from field plots were used for deoxynivalenol (DON) determination. A total of 328 polymorphic microsatellite loci were used to construct a genetic linkage map in this population and together these data were used to identify QTL controlling FHB resistance, accumulation of DON, and agronomic and spike morphology traits. The analysis identified QTL for different types of FHB resistance in four intervals on chromosomes 2DL, 3BS, and 4B. The QTLs on 4B and 3BS proximal to the centromere are novel and not reported elsewhere. QTL controlling accumulation of DON independent of FHB resistance were located on chromosomes 2DS and 5AS. Lines carrying FHB resistance alleles on 2DL and 3BS showed a 32% decrease in disease spread after single-floret injection. Lines carrying FHB resistance alleles on 3BS and 4B showed a 27% decrease from the mean in field infection. Finally, lines carrying favourable alleles on 3BS and 5AS, showed a 17% reduction in DON accumulation. The results support a polygenic and quantitative mode of inheritance and report novel FHB resistance loci. The data also suggest that resistance to FHB infection and DON accumulation may be controlled, in part, by independent loci and (or) genes.     

Fusarium graminearum gene deletion mutants map1 and tri5 reveal similarities and differences in the pathogenicity requirements to cause disease on Arabidopsis and wheat floral tissue

The Ascomycete pathogen Fusarium graminearum can infect all cereal species and lower grain yield, quality and safety. The fungus can also cause disease on Arabidopsis thaliana. In this study, the disease-causing ability of two F. graminearum mutants was analysed to further explore the parallels between the wheat (Triticum aestivum) and Arabidopsis floral pathosystems. Wild-type F. graminearum (strain PH-1) and two isogenic transformants lacking either the mitogen-activated protein kinase MAP1 gene or the trichodiene synthase TRI5 gene were individually spray- or point-inoculated onto Arabidopsis and wheat floral tissue. Disease development was quantitatively assessed both macroscopically and microscopically and deoxynivalenol (DON) mycotoxin concentrations determined by enzyme-linked immunosorbent assay (ELISA). Wild-type strain inoculations caused high levels of disease in both plant species and significant DON production. The map1 mutant caused minimal disease and DON accumulation in both hosts. The tri5 mutant, which is unable to produce DON, exhibited reduced pathogenicity on wheat ears, causing only discrete eye-shaped lesions on spikelets which failed to infect the rachis. By contrast, the tri5 mutant retained full pathogenicity on Arabidopsis floral tissue. This study reveals that DON mycotoxin production is not required for F. graminearum to colonize Arabidopsis floral tissue.     

Differential expression of proteins in response to the interaction between the pathogen Fusarium graminearum and its host, Hordeum vulgare

Using proteomic techniques, a study aimed at isolating and identifying proteins associated with resistance to fusarium head blight (FHB) was conducted on six barley genotypes of varying resistance. At anthesis, barley spikelets were point inoculated with Fusarium graminearum macroconidial suspensions or mock inoculum. In total, 43 acidic protein spots out of 600 were detected 3 days postinoculation to be differentially expressed due to FHB and were identified. Identification of proteins responsive to FHB included those associated with oxidative burst and oxidative stress response, such as malate dehydrogenase and peroxidases, and pathogenesis-related (PR). An increase in abundance of PR-3 or PR-5 could be associated with the resistant genotypes CI4196, Svansota, and Harbin, as well as the intermediate resistant genotype CDC Bold. On the contrary, the susceptible genotype Stander showed a decrease in abundance of these acidic PR-proteins. In the susceptible and intermediate resistant genotypes Stander and CDC Bold, as well as CI4196, the increased abundance of proteins associated with an oxidative response might have prepared the terrain for saprophytic fungal invasion. On the contrary, in the resistant sources Harbin and Svansota we did not observed change in abundance of these proteins. Not a single significant change in acidic protein abundance could be detected in Chevron. Three distinct response patterns are reported from these six barley genotypes.     

Detection of QTL linked to Fusarium head blight resistance in Sumai 3-derived North Dakota bread wheat lines

During the past decade Fusarium head blight (FHB) caused by Fusarium graminearum Schwabe has resulted in severe grain yield and quality losses of wheat (Triticum aestivum L.) in the Northern Great Plains of the U.S. Given the complexity of breeding for FHB resistance, molecular markers associated with this trait will be valuable in accelerating efforts to breed resistant cultivars. The objective of this study was to identify molecular markers linked to quantitative trait loci (QTL) for FHB resistance in wheat using a set of lines obtained by several cycles of crossing to North Dakota adapted genotypes, which derived their resistance from cv. Sumai 3. Microsatellite markers spanning the wheat genome were used to screen parents and derived lines. Polymorphisms for parental alleles were compared to disease scores for Type II resistance. The probability of linkage between markers and introgressed resistance genes was calculated using a binomial probability formula based on the assumption that a molecular marker at a specific distance from the introgressed gene, in a near-isogenic line (NIL), will carry the donor-parent allele as a function of the distance between marker and gene and the number of backcrosses/selfs performed in deriving the NIL. Microsatellite loci Xgwm533 and Xgwm274 were significantly associated with QTL for FHB resistance.