Overexpression of defense response genes in transgenic wheat enhances resistance to Fusarium head blight

Fusarium head blight (FHB) of wheat, caused by Fusarium graminearum and other Fusarium species, is a major disease problem for wheat production worldwide. To combat this problem, large-scale breeding efforts have been established. Although progress has been made through standard breeding approaches, the level of resistance attained is insufficient to withstand epidemic conditions. Genetic engineering provides an alternative approach to enhance the level of resistance. Many defense response genes are induced in wheat during F. graminearum infection and may play a role in reducing FHB. The objectives of this study were (1) to develop transgenic wheat overexpressing the defense response genes α-1-purothionin, thaumatin-like protein 1 (tlp-1), and β-1,3-glucanase; and (2) to test the resultant transgenic wheat lines against F. graminearum infection under greenhouse and field conditions. Using the wheat cultivar Bobwhite, we developed one, two, and four lines carrying the α-1-purothionin, tlp-1, and β-1,3-glucanase transgenes, respectively, that had statistically significant reductions in FHB severity in greenhouse evaluations. We tested these seven transgenic lines under field conditions for percent FHB disease severity, deoxynivalenol (DON) mycotoxin accumulation, and percent visually scabby kernels (VSK). Six of the seven lines differed from the nontransgenic parental Bobwhite line for at least one of the disease traits. A β-1,3-glucanase transgenic line had enhanced resistance, showing lower FHB severity, DON concentration, and percent VSK compared to Bobwhite. Taken together, the results showed that overexpression of defense response genes in wheat could enhance the FHB resistance in both greenhouse and field conditions.     

Susceptibility to <Emphasis Type="Italic">Fusarium </Emphasis>head blight is associated with the <Emphasis Type="Italic">Rht-D1b</Emphasis> semi-dwarfing allele in wheat

Fusarium head blight (FHB) is an important disease of wheat worldwide. The cultivar Spark is more resistant than most other UK winter wheat varieties but the genetic basis for this is not known. A mapping population from a cross between Spark and the FHB susceptible variety Rialto was used to identify quantitative trait loci (QTL) associated with resistance. QTL analysis across environments revealed nine QTL for FHB resistance and four QTL for plant height (PH). One FHB QTL was coincident with the Rht-1D locus and accounted for up to 51% of the phenotypic variance. The enhanced FHB susceptibility associated with Rht-D1b is not an effect of PH per se as other QTL for height segregating in this population have no influence on susceptibility. Experiments with near-isogenic lines supported the association between susceptibility and the Rht-D1b allele conferring the semi-dwarf habit. Our results demonstrate that lines carrying the Rht-1Db semi-dwarfing allele are compromised in resistance to initial infection (type I resistance) while being unaffected in resistance to spread within the spike (type II resistance).     

Resistance against Fusarium Head Blight in Transgenic Wheat Plants Expressing the ScNPR1 gene

Fusarium head blight (FHB) is a severe global wheat disease that may cause severe yield losses, especially during epidemic years. Transforming the regulatory genes in the metabolic pathways of disease resistance into wheat via transgenic methods is one way to improve resistance to FHB. ScNPR1 (Secale cereale‐NPR1), a regulatory gene for systemic acquired resistance (SAR), was isolated from S. cereale cv Jingzhouheimai and transformed into the moderately FHB‐susceptible wheat variety Ningmai 13. RT‐PCR analysis indicated that the ScNPR1 gene was stably expressed in transgenic plants. An evaluation of the resistance to FHB revealed that six ScNPR1 transgenic lines (NP1, NP2, NP3, NP4, NP5 and NP6) exhibited significantly higher FHB resistance than the wild‐type wheat Ningmai 13 and the null‐segregated plants. The expression of pathogenesis‐related (PR) genes after Fusarium graminearum inoculation was earlier or higher than those in the wild‐type variety Ningmai 13. The high expression in the early stages of PR genes should account for the enhanced FHB resistance in the transgenic lines. Our results suggest that overexpression of ScNPR1 could be used to improve FHB resistance in wheat.     

Identification of QTLs for resistance to Fusarium head blight, DON accumulation and associated traits in the winter wheat variety Arina

Fusarium head blight (FHB) of wheat has become a serious threat to wheat crops in numerous countries. In addition to loss of yield and quality, this disease is of primary importance because of the contamination of grain with mycotoxins such as deoxynivalenol (DON). The Swiss winter cultivar Arina possesses significant resistance to FHB. The objective of this study was to map quantitative trait loci (QTL) for resistance to FHB, DON accumulation and associated traits in grain in a double haploid (DH) population from a cross between Arina and the FHB susceptible UK variety Riband. FHB resistance was assessed in five trials across different years and locations. Ten QTL for resistance to FHB or associated traits were detected across the trials, with QTL derived from both parents. Very few of the QTL detected in this study were coincident with those reported by authors of two other studies of FHB resistance in Arina. It is concluded that the FHB resistance of Arina, like that of the other European winter wheat varieties studied to date, is conferred by several genes of moderate effect making it difficult to exploit in marker-assisted selection breeding programmes. The most significant and stable QTL for FHB resistance was on chromosome 4D and co-localised with the Rht–D1 locus for height. This association appears to be due to linkage of deleterious genes to the Rht-D1b (Rht2) semi-dwarfing allele rather than differences in height per se. This association may compromise efforts to enhance FHB resistance in breeding programmes using germplasm containing this allele.     

Genetic dissection of a major Fusarium head blight QTL in tetraploid wheat

The devastating effect of Fusarium head blight (FHB) caused by Fusarium graminearum has led to significant financial losses across the Upper Midwest of the USA. These losses have spurred the need for research in biological, chemical, and genetic control methods for this disease. To date, most of the research on FHB resistance has concentrated on hexaploid wheat (Triticum aestivum L.) lines originating from China. Other sources of resistance to FHB would be desirable. One other source of resistance for both hexaploid wheat and tetraploid durum wheat (T. turgidum L. var. durum) is the wild tetraploid, T. turgidum L. var. dicoccoides (T. dicoccoides). Previous analysis of the `Langdon'-T. dicoccoides chromosome substitution lines, LDN(Dic), indicated that the chromosome 3A substitution line expresses moderate levels of resistance to FHB. LDN(Dic-3A) recombinant inbred chromosome lines (RICL) were used to generate a linkage map of chromosome 3A with 19 molecular markers spanning a distance of 155.2 cM. The individual RICL and controls were screened for their FHB phenotype in two greenhouse seasons. Analysis of 83 RICL identified a single major quantitative trait locus, Qfhs.ndsu-3AS, that explains 37% of the phenotypic or 55% of the genetic variation for FHB resistance. A microsatellite locus, Xgwm2, is tightly linked to the highest point of the QTL peak. A region of the LDN (Dic-3A) chromosome associated with the QTL for FHB resistance encompasses a 29.3 cM region from Xmwg14 to Xbcd828.     

The evaluation of FHB resistance QTLs introgressed into elite Canadian spring wheat germplasm

FHB resistance QTL alleles from Nyuubai, Sumai-3, and Wuhan-1 were evaluated for their effect on Fusarium head blight (FHB) index, Fusarium damaged kernels (FDK), deoxynivalenol (DON) accumulation, plant height, anthesis date, and numerous grain quality traits in three elite Canadian spring wheat backgrounds. The three FHB resistance parameters were negatively correlated with plant height in the three populations. The Wuhan-1 4B resistance allele was the most effective resistance allele but was associated with a 9.3 cm increase in plant height. The Wuhan-1 2D, Nyuubai 3BSc, Sumai-3 3BSc, Nyuubai 5AS, and Sumai-3 5AS alleles were also effective FHB resistance alleles in these populations. The Nyuubai and Sumai-3 3BS alleles were the least effective of the FHB resistance alleles in the FHB nursery tests. The Sumai-3 5AS resistance allele was significantly associated with reduced grain protein content, while the same trend was observed for the Nyuubai 5AS resistance allele but was not significant. FHB resistance tended to increase with more FHB resistance alleles introgressed into the elite genetic background, which suggested that marker-assisted selection (MAS) will prove useful for improving FHB resistance in Canadian germplasm.     

Mapping of Fhb2 on chromosome 6BS: a gene controlling Fusarium head blight field resistance in bread wheat (Triticum aestivum L.)

Fusarium head blight (FHB) is one of the most important fungal wheat diseases worldwide. Understanding the genetics of FHB resistance is key to facilitate the introgression of different FHB resistance genes into adapted wheat. The objective of this project was to study the FHB resistance QTL on chromosome 6B, quantify the phenotypic variation, and qualitatively map the resistance gene as a Mendelian factor. The FHB resistant parent BW278 (AC Domain*2/Sumai 3) was used as the source of the resistance allele. A large recombinant inbred line (RIL) mapping population was developed from the cross BW278/AC Foremost. The population segregated for three known FHB resistance QTL located on chromosomes 3BSc, 5A, and 6B. Molecular markers on chromosome 6B (WMC104, WMC397, GWM219), 5A (GWM154, GWM304, WMC415), and 3BS (WMC78, GWM566, WMC527) were amplified on approximately 1,440 F_2:7 RILs. The marker information was used to select 89 RILs that were fixed homozygous susceptible for the 3BSc and 5A FHB QTLs and were recombinant in the 6B interval. Disease response was evaluated on 89 RILs and parental checks in the greenhouse and field nurseries. Dual floret injection (DFI) was used in greenhouse trials to evaluate disease severity (DS). Macroconidial spray inoculations were used in field nurseries conducted at two locations in southern Manitoba (Carman and Glenlea) over two years 2003 and 2004, to evaluate disease incidence, disease severity, visual rating index, and Fusarium-damaged kernels. The phenotypic distribution for all five-disease infection measurements was bimodal, with lines resembling either the resistant or susceptible checks and parents. All of the four field traits for FHB resistance mapped qualitatively to a coincident position on chromosome 6BS, flanked by GWM133 and GWM644, and is named Fhb2. The greenhouse-DS trait mapped 2 cM distal to Fhb2. Qualitative mapping of Fhb2 in wheat provides tightly linked markers that can reduce linkage drag associated with marker assisted selection of Fhb2 and aid the pyramiding of different resistance loci for wheat improvement.     

Saturation and comparative mapping of a major Fusarium head blight resistance QTL in tetraploid wheat

Fusarium head blight (FHB) is a devastating disease of cultivated wheat worldwide. Partial resistance to FHB has been identified in common wheat (Triticum aestivum L.). However, sources of effective FHB resistance have not been found in durum wheat (T. turgidum L. var. durum). A major FHB resistance quantitative trait loci (QTL), Qfhs.ndsu-3AS, was identified on chromosome 3A of T. dicoccoides, a wild relative of durum wheat. Here, we saturated the genomic region containing the QTL using EST-derived target region amplified polymorphism (TRAP), sequence tagged site (STS), and simple sequence repeat (SSR) markers. A total of 45 new molecular marker loci were detected on chromosome 3A and the resulting linkage map consisted of 55 markers spanning a genetic distance of 277.2 cM. Qfhs.ndsu-3AS was positioned within a chromosomal interval of 11.5 cM and is flanked by the TRAP marker loci, Xfcp401 and Xfcp397.2. The average map distance between the marker loci within this QTL region was reduced from 4.9 cM in the previous study to 3.5 cM in the present study. Comparative mapping indicated that Qfhs.ndsu-3AS is not homoeologous to Qfhs.ndsu-3BS, a major FHB QTL derived from the common wheat cultivar Sumai 3. These results facilitate our efforts toward map-based cloning of Qfhs.ndsu-3AS and utilization of this QTL in durum wheat breeding via marker-assisted selection.     

Fusarium head blight biological control with Lysobacter enzymogenes strain C3

Fusarium head blight (FHB), caused by Fusarium graminearum (= Gibberella zeae), is a destructive disease of wheat for which biological controls are needed. Lysobacter enzymogenes strain C3, a bacterial antagonist of fungal pathogens via lytic enzymes and induced resistance, was evaluated in this study for control of FHB. In greenhouse experiments, chitin broth cultures of C3 reduced FHB severity to <10% infected spikelets as compared to >80% severity in the controls in some experiments. C3 broth cultures heated to inactivate cells and lytic enzymes, but retaining the elicitor factor for induced resistance, also were effective in reducing FHB severity, suggesting induced resistance is one mechanism of action. C3 broth cultures also were effective when applied in highly diluted form and when applied 1 week prior to pathogen inoculation. When applied to 8 cultivars of hard red spring wheat in the greenhouse, C3 treatments reduced FHB in 5 cultivars but not in the others. These findings also are consistent with induced resistance. Protection offered by C3 treatments, however, was not systemic and required that C3 be applied uniformly to all susceptible florets. Field tests were conducted in South Dakota and Nebraska to evaluate the efficacy of C3 chitin broth cultures in spring and winter wheat, respectively. In experiments involving two hard red spring wheat cultivars, treatment with C3 reduced FHB severity in ‘Russ’ but not in ‘Ingot’. In three other field experiments comparing C3, the fungicide tebuconazole, and the combination of C3 and tebuconazole, treatments with the bacterial culture alone and the fungicide alone were inconsistent across experiments, each treatment being ineffective in controlling FHB in one experiment. The biocontrol agent–fungicide combination was more consistently effective, reducing FHB incidence or severity in all three experiments. Thus, the potential for using L. enzymogenes C3 as a biological control agent for FHB was demonstrated along with a number of factors that might affect control efficacy in the field.     

Molecular mapping of <Emphasis Type="Italic">Thinopyrum</Emphasis>-derived Fusarium head blight resistance in common wheat

Resistance to Fusarium head blight (FHB) caused by Fusarium graminearum Schwabe in wheat (Triticum aestivum L.) was identified in disomic chromosome substitution and translocation lines, into which chromosome 7el₂ had been introgressed from wheatgrass, Thinopyrum ponticum. In this study, two chromosome substitution lines with different origins (designated as el₁ and el₂) and with different reactions to infection by F. graminearum were crossed to develop a segregating mapping population. The objectives of this study were to determine the effectiveness of this type II resistance and map it on chromosome 7el₂. Type II resistance to FHB was characterized in the F₂, F_2:3 families, F_4:5 plants and F_5:6 recombinant inbred lines developed by single-seed descent; and the population was characterized in the F₂ and F₅ with DNA markers along the long arm of 7el. Composite interval mapping revealed a FHB resistance QTL, designated Qfhs.pur-7EL, located in the distal region of the long arm of 7el₂ and delimited with flanking markers XBE445653 and Xcfa2240. Additive effects of Qfhs.pur-7EL reduced the number of diseased spikelets per spike following inoculation of one floret in four experiments by 1.5–2.6 and explained 15.1–32.5% of the phenotypic variation in the populations. Several STS-derived and EST-derived PCR or CAPS markers were developed in this chromosomal region, and showed the specificity of 7el₂ compared to an array of wheat lines possessing other sources of FHB resistance. These markers are useful in an effort to shorten the chromosome segment of 7el₂ and to use for marker-assisted introgression of this resistance into wheat.     

Selection and evaluation of the potential of choline-metabolizing microbial strains to reduce Fusarium head blight

Choline and betaine are found in wheat flower tissues and have been implicated in stimulating hyphal growth of the primary causal agent of Fusarium head blight (FHB), Gibberella zeae. Choline metabolizing strains (CMS) from wheat anthers may therefore be a useful source of antagonists of G. zeae. One-hundred twenty-three of 738 microbial strains that were recovered from wheat anthers collected from plants grown in Illinois and Ohio were CMS as determined by growth in a liquid medium containing choline as a sole carbon and nitrogen source and a colorimetric, choline oxidase-based assay of culture filtrate. Thirty-one out of 123 CMS reduced FHB disease severity by at least 25% in greenhouse tests on wheat and 17 reduced FHB severity by at least 50%. All five CMS selected for field testing in 2003 reduced disease severity compared to the untreated check at both field locations on moderately resistant cultivar Freedom. Freedom wheat treated with Pseudomonas sp. AS 64.4 had 63% and 46% less FHB severity than untreated wheat at the two sites. Three of five CMS reduced severity at both locations on susceptible cultivar Pioneer Brand 2545. Disease control was comparable to that obtained using the fungicide Folicur 3.6F. Selection of wheat anther colonists for ability to utilize choline as a sole carbon and nitrogen source has utility as a screening tool in the search for efficacious antagonists of G. zeae although choline utilization does not insure that an isolate will be an effective biocontrol agent against Fusarium head blight.     

Influence of localities and winter wheat cultivars on deoxynivalenol accumulation and disease damage by <Emphasis Type="Italic">Fusarium culmorum</Emphasis>

Toxin B — trichothecene deoxynivalenol (DON) is the most frequent Fusarium mycotoxin in Fusarium head blight (FHB) disease produced by Fusarium fungi. Thirty-one samples of naturally cultivated winter wheat were collected from different localities in Slovakia and evaluated for DON content, and after an artificial inoculation twelve of winter wheat cultivars were evaluated for FHB, fusarium damaged kernels (FDK) and DON content (resistance Type I and II) during two years. Plants were inoculated at anthesis with a conidial suspension of Fusarium culmorum (W. G. Smith) Sacc. The highest mean contents of DON 1.641 ppm were found in produced potato region (PPR) and 1.654 ppm in produced sugar beet region (PSBR). A positive correlation was found between DON content and rainfall, and a negative correlation was found between content of DON and temperature. Lower positive correlations were found between the contents of DON in 2003 and 2004 in the resistance Type I and Type II in twelve artificially infected cultivars. The significant positive correlations in content of DON were found between resistance Type I and Type II in the years 2003 and 2004. The lowest content of DON was found in the cultivars Alka, Malyska and the highest one in the cultivars Vanda and Boka. The positive correlation between the content of DON and FDK (in %) in head (average 2003 and 2004 years) from artificially infected and analysed cultivars was statistically significant in both resistances Type I and Type II.     

Host‐induced gene silencing of an essential chitin synthase gene confers durable resistance to Fusarium head blight and seedling blight in wheat

Fusarium head blight (FHB) and Fusarium seedling blight (FSB) of wheat, caused by Fusarium pathogens, are devastating diseases worldwide. We report the expression of RNA interference (RNAi) sequences derived from an essential Fusarium graminearum (Fg) virulence gene, chitin synthase (Chs) 3b, as a method to enhance resistance of wheat plants to fungal pathogens. Deletion of Chs3b was lethal to Fg; disruption of the other Chs gene family members generated knockout mutants with diverse impacts on Fg. Comparative expression analyses revealed that among the Chs gene family members, Chs3b had the highest expression levels during Fg colonization of wheat. Three hairpin RNAi constructs corresponding to the different regions of Chs3b were found to silence Chs3b in transgenic Fg strains. Co‐expression of these three RNAi constructs in two independent elite wheat cultivar transgenic lines conferred high levels of stable, consistent resistance (combined type I and II resistance) to both FHB and FSB throughout the T₃ to T₅ generations. Confocal microscopy revealed profoundly restricted mycelia in Fg‐infected transgenic wheat plants. Presence of the three specific short interfering RNAs in transgenic wheat plants was confirmed by Northern blotting, and these RNAs efficiently down‐regulated Chs3b in the colonizing Fusarium pathogens on wheat seedlings and spikes. Our results demonstrate that host‐induced gene silencing of an essential fungal chitin synthase gene is an effective strategy for enhancing resistance in crop plants under field test conditions.     

Utilization of chemical inducers of resistance and Cryptococcus flavescens OH 182.9 to reduce Fusarium head blight under greenhouse conditions

Four chemicals [salicylic acid (SA), sodium salt of salicylic acid (NaSA), isonicotinic acid (INA), and DL-β-amino-n-butyric acid (BABA)] and the yeast antagonist Cryptococcus flavescens (=C. nodaensis nomen nudum) OH 182.9 were evaluated separately or together for the ability to reduce Fusarium head blight (FHB) of wheat in the greenhouse. When sprayed onto wheat heads at 3 days prior to pathogen challenge with Gibberella zeae, NaSA and INA at 10 mM significantly reduced FHB severity compared to the non-treated disease control. Applied at concentrations of 1 and 5 mM at 3 days before pathogen challenge, NaSA or INA in combination with OH 182.9 did not significantly reduce FHB severity compared to either treatment alone, though the lowest disease severity values frequently were associated with the combination treatments. When sprayed onto wheat heads just beginning to emerge from boot at 10 days prior to pathogen inoculation, NaSA, INA, and BABA at 1 mM significantly reduced FHB severity indicating that induced systemic resistance was at least partially responsible for the reduction of FHB disease. Induced FHB resistance was achieved by treating wheat with INA at concentrations as low as 0.1 mM. In only one instance was 100-kernel weight affected by any chemical or combination of chemicals with OH 182.9 treatment. Data from our studies in the greenhouse suggest that chemical inducers can induce resistance in wheat against FHB, and that further efforts are warranted to explore the potential of improved control of FHB disease by incorporating chemical inducers with the FHB biocontrol agent OH 182.9.     

Recognition of glycoside hydrolase 12 proteins by the immune receptor RXEG1 confers Fusarium head blight resistance in wheat

Fusarium head blight (FHB), caused by Fusarium graminearum, is a devastating disease in wheat (Triticum aestivum) that results in substantial yield losses and mycotoxin contamination. Reliable genetic resources for FHB resistance in wheat are lacking. In this study, we characterized glycoside hydrolase 12 (GH12) family proteins secreted by F. graminearum. We established that two GH12 proteins, Fg05851 and Fg11037, have functionally redundant roles in F. graminearum colonization of wheat. Furthermore, we determined that the GH12 proteins Fg05851 and Fg11037 are recognized by the leucine-rich-repeat receptor-like protein RXEG1 in the dicot Nicotiana benthamiana. Heterologous expression of RXEG1 conferred wheat responsiveness to Fg05851 and Fg11037, enhanced wheat resistance to F. graminearum and reduced levels of the mycotoxin deoxynivalenol in wheat grains in an Fg05851/Fg11037-dependent manner. In the RXEG1 transgenic lines, genes related to pattern-triggered plant immunity, salicylic acid, jasmonic acid, and anti-oxidative homeostasis signalling pathways were upregulated during F. graminearum infection. However, the expression of these genes was not significantly changed during infection by the deletion mutant ΔFg05851/Fg11037, suggesting that the recognition of Fg05851/Fg11037 by RXEG1 triggered plant resistance against FHB. Moreover, introducing RXEG1 into three other different wheat cultivars via crossing also conferred resistance to F. graminearum. Expression of RXEG1 did not have obvious deleterious effects on plant growth and development in wheat. Our study reveals that N. benthamiana RXEG1 remains effective when transferred into wheat, a monocot, which in turn suggests that engineering wheat with interfamily plant immune receptor transgenes is a viable strategy for increasing resistance to FHB.     

QTL associated with Fusarium head blight resistance in the soft red winter wheat Ernie

Fusarium head blight (FHB), mainly caused by Fusarium graminearum Schwabe [telomorph: Gibberella zeae Schw. (Petch)], is an increasingly important disease of wheat (Triticum aestivum L.). Host-plant resistance provides the best hope for reducing economic losses associated with FHB, but new sources of resistance are limited. The moderately resistant winter wheat cultivar, Ernie, may provide a source of resistance that differs from Sumai 3 but these genes have not been mapped. Also hindering resistance breeding may be associations of resistance with agronomic traits such as late maturity that may be undesirable in some production environments. This research was conducted to identify QTL associated with type II FHB resistance (FHB severity, FHBS), and to determine if they are associated with days to anthesis (DTA), number of spikelets (NOS), and the presence/absence of awns. Two hundred and forty-three F₈ recombinant inbred lines from a cross between the resistant cultivar, Ernie and susceptible parent, MO 94-317 were phenotyped for type II FHB resistance using point inoculation in the greenhouse during 2002 and 2003. Genetic linkage maps were constructed using 94 simple sequence repeat (SSR) and 146 amplified fragment length polymorphic (AFLP) markers. Over years four QTL regions on chromosomes 2B, 3B, 4BL and 5A were consistently associated with FHB resistance. These QTL explained 43.3% of the phenotypic variation in FHBS. Major QTL conditioning DTA and NOS were identified on chromosome 2D. Neither the QTL associated with DTA and NOS nor the presence/absence of awns were associated with FHB resistance in Ernie. Our results suggest that the FHB resistance in Ernie appears to differ from that in Sumai 3, thus pyramiding the QTL in Ernie with those from Sumai 3 could result in enhanced levels of FHB resistance in wheat.     

Osmotic stress adaptation, compatible solutes accumulation and biocontrol efficacy of two potential biocontrol agents on Fusarium head blight in wheat

Fusarium head blight (FHB) caused by Gibberella zeae (anamorph = Fusarium graminearum) is a devastating disease that causes extensive yield and quality losses to wheat in humid and semi-humid regions of the world. Biological control has been demonstrated to be effective under laboratory conditions but a few biocontrol products have been effective under field conditions. The improvement in the physiological quality of biocontrol agents may improve survival under field conditions, and therefore, enhance biocontrol activity. Bacillus subtilis RC 218 and Brevibacillus sp. RC 263 were isolated from wheat anthers and showed significant effect on control of FHB under greenhouse assays. This study showed the effect of water availability measured as water activity (a_W) using a growth medium modified with NaCl, glycerol and glucose on: (i) osmotic stress tolerance, (ii) viability in modified liquid medium, (iii) quantitative intracellular accumulation of betaine and ectoine and (iv) the biocontrol efficacy of the physiologically improved agents. Viability of B. subtilis RC 218 in NaCl modified media was similar to the control. Brevibacillus sp. RC 263 showed a limited adaptation to growth in osmotic stress. Betaine was detected in high levels in modified cells but ectoine accumulation was similar to the control cells. Biocontrol activity was studied in greenhouse assays on wheat inoculated at anthesis period with F. graminearum RC 276. Treatments with modified bacteria reduced disease severity from 60% for the control to below 20%. The physiological improvement of biocontrol agents could be an effective strategy to enhance stress tolerance and biocontrol activity under fluctuating environmental conditions.     

Transgenic rice plants expressing trichothecene 3-<Emphasis Type="Italic">O</Emphasis>-acetyltransferase show resistance to the <Emphasis Type="Italic">Fusarium</Emphasis> phytotoxin deoxynivalenol

Fusarium head blight (FHB) is a devastating disease of small grain cereal crops caused by the necrotrophic pathogen Fusarium graminearum and Fusarium culmorum. These fungi produce the trichothecene mycotoxin deoxynivalenol (DON) and its derivatives, which enhance the disease development during their interactions with host plants. For the self-protection, the trichothecene producer Fusarium species have Tri101 encoding trichothecene 3-O-acetyltransferase. Although transgenic expression of Tri101 significantly reduced inhibitory action of DON on tobacco plants, there are several conflicting observations regarding the phytotoxicity of 3-acetyldeoxynivalenol (3-ADON) to cereal plants; 3-ADON was reported to be highly phytotoxic to wheat at low concentrations. To examine whether cereal plants show sufficient resistance to 3-ADON, we generated transgenic rice plants with stable expression and inheritance of Tri101. While root growth of wild-type rice plants was severely inhibited by DON in the medium, this fungal toxin was not phytotoxic to the transgenic lines that showed trichothecene 3-O-acetylation activity. This is the first report demonstrating the DON acetylase activity and DON-resistant phenotype of cereal plants expressing the fungal gene. S. Ohsato and T. Ochiai-Fukuda should be considered as joint first authors.     

Targeting the pattern-triggered immunity pathway to enhance resistance to Fusarium graminearum

Fusarium head blight (FHB) is a disease of the floral tissues of wheat and barley for which highly resistant varieties are not available. Thus, there is a need to identify genes/mechanisms that can be targeted for the control of this devastating disease. Fusarium graminearum is the primary causal agent of FHB in North America. In addition, it also causes Fusarium seedling blight. Fusarium graminearum can also cause disease in the model plant Arabidopsis thaliana. The Arabidopsis–F. graminearum pathosystem has facilitated the identification of targets for the control of disease caused by this fungus. Here, we show that resistance against F. graminearum can be enhanced by flg22, a bacterial microbe-associated molecular pattern (MAMP). flg22-induced resistance in Arabidopsis requires its cognate pattern recognition receptor (PRR) FLS2, and is accompanied by the up-regulation of WRKY29. The expression of WRKY29, which is associated with pattern-triggered immunity (PTI), is also induced in response to F. graminearum infection. Furthermore, WRKY29 is required for basal resistance as well as flg22-induced resistance to F. graminearum. Moreover, constitutive expression of WRKY29 in Arabidopsis enhances disease resistance. The PTI pathway is also activated in response to F. graminearum infection of wheat. Furthermore, flg22 application and ectopic expression of WRKY29 enhance FHB resistance in wheat. Thus, we conclude that the PTI pathway provides a target for the control of FHB in wheat. We further show that the ectopic expression of WRKY29 in wheat results in shorter stature and early heading time, traits that are important to wheat breeding.     

Marker Assisted Selection of Fusarium Head Blight Resistance Genes in Two Doubled­ Haploid Populations of Wheat

Fusarium head blight, caused primarily by Fusarium graminearum, is the most important wheat disease in Canada causing both grain yield and quality losses. Selection for resistance to Fusarium head blight in breeding programs has been difficult because of the complex inheritance of resistance and the environmental effect on disease development and expression. The present study was conducted to examine microsatellite markers associated with resistance to Fusarium head blight and evaluate the effectiveness of these microsatellite markers in selecting for resistance to Fusarium head blight in two doubled-haploid populations segregating for Sumai 3-derived resistance genes. Both doubled-haploid populations were evaluated for resistance to Fusarium head blight by inoculation with F. graminearum in the greenhouse. Eight microsatellite markers from chromosomes 3BS, 6B and 5AL were applied to both doubled-haploid populations. The most significant microsatellite markers were found on the short arm of chromosome 3B, explaining 12% and 36% of phenotypic variation for resistance in the DH181/AC Foremost and AC Foremost/93FHB 21 doubled-haploid populations, respectively. Another important microsatellite marker, gwm644 on 6B, explained 21 % of the phenotypic variation for resistance to Fusarium head blight in the DH181/AC Foremost doubled-haploid population. There was a general lack of marker polymorphism on 5AL for the parents used in this study. Microsatellite markers on chromosome 3BS in addition to microsatellite markers on 6B have the potential for accelerating the development of wheat cultivars with improved Fusarium head blight resistance through the use of marker-assisted selection.