Deliverables
Our studies highlight several new candidate genes involved in the wheat response to both DON and F. graminearum [retrotransposons (Erika LTR and Romani PP), a class III peroxidase (POX), a structure-specific recognition protein (SSRP1) and a basic leucine zipper transcription factor (bZIP)]. While many of the transcripts identified in this research may play a role in combating stress, so far only bZIP any potential link with the inheritance of QTL 3BS and the QTL3BS-associated DON tolerance. bZIP comprises a large family of transcription factors. The closest homologs of the DON responsive bZIP in GenBank are wheat and rice lip19 genes encoding basic leucine zipper proteins.
We have characterized the FHB resistance of a wheat Screening Nursery with 126 genotypes from diverse sources (mainly winter wheat and some spring wheat genotypes) which were described as moderately to highly resistant against FHB (by own results, in the literature or by pers. comm.), together with a few susceptible checks and breeding lines of the participating breeding companies.
The material was evaluated for FHB resistance in well-replicated experiments during three seasons at five locations across Europe (in total 14 different year by location combinations). The lines were tested using artificial inoculations with local Fusarium strains and local methodology by spraying the Fusarium inoculum directly on the heads at anthesis or by the distribution of Fusarium plant debris on the soil surface. In this way this nursery of wheat lines was well characterized for FHB resistance in direct comparisons.
Severity was assessed. This disease parameter is a measure for the overall FHB resistance of the investigated lines. It is the result of a combination of Type I (resistance against penetration of the ear) and Type II resistance (resistance against colonization, i.e. growth of the fungus in the ear after infection took place). Incidence primarily reflects Type I resistance, whereas both spread and wilting measure Type II resistance. All data were analyzed and are available for further distribution. Highly significant differences between FHB resistance of the tested lines were detected. Type I and Type II resistance seems not to be coupled.
The data will be published. In addition, this set of genotypes (excluding the breeding lines) is made available to any interested breeding company also after the end of the project.
Moreover, the most promising lines are used by the participating private breeders immediately in their crossing programs as novel sources for FHB resistance. They can also be used by any third party. Also the resistance data of the individual lines are available freely.
This will not only allow any interested breeder to use the resistant lines for breeding, but also to improve the artificial inoculation techniques used. The resistance data obtained by any third party can be compared with the resistance data from our consortium. Our resistance data can be regarded as the true resistance level of these genotypes because of the intensive phenotyping we did.
In the screen for DON-resistance conferring wheat cDNAs we found 18 plasmids containing 8 different types of inserts. The molecular mechanism responsible for increased resistance is suggested by only a few of the sequences. Potentially such DON-resistance conferring cDNAs could be relevant for plant biotechnology if such a phenotype can also be obtained in transgenic (model) plants.
Based on previous knowledge the aim was to identify DNA markers closely linked to two important Fusarium head blight resistance genes derived from the spring wheat line CM-82036. We developed two high resolution mapping populations of about 1000 F2 derived recombinant inbred lines. We selected recombinant lines for the putative QTL regions and analysed these in depth with molecular markers and for their Fusarium resistance traits in replicated field experiments.
The high-resolution mapping led to the identification of close SSR markers for Qfhs.ndsu-3BS: Barc133 and Barc147. Qfhs.ifa-5A resides in an area of reduced recombination around the centromere of chromosome 5A. This reduced recombination area on 5A is a general feature as it was also found in several independent F2 populations. At least 8 SSR markers were closely linked in this area and any of these may be used for breeding purposes. Genetic diversity analysis has given interesting insight in the relation between and among lines from different breeding programs and germplasm sources.
We have developed yeast strains that are sensitive to trichothecenes and have a conditional RPL3 gene (expressed on galactose medium and therfore viable, not transcribed on glucose medium). Such strains can be transformed with plant cDNAs encoding wildtype or mutant alleles of RPL3, and the ability of the transgene to replace the yeast gene functionally and its toxin resistance properties can be phenotypically detected.
We have generated a double haploid population of 114 lines from a cross between the Fusarium head blight (FHB) resistant variety Arina and the FHB susceptible variety Riband. We have developed a genetic map for this cross using a combination of SSR and AFLP molecular markers. In collaboration with our partners in this project we have carried out a series of trials to determine the FHB resistance level of each line.
By combining the genetic information for each line, the distribution of markers across the genome (genetic map) and the disease scoring data we have established the location of the loci that contribute most to the difference in FHB resistance between Arina and Riband.
This information includes the identity of the molecular markers associated with each resistance locus. These markers can be used within plant breeding programmes to identify lines with high or low resistance to FHB. Using these markers should enable efficient selection of lines that carry significant FHB resistance.
At present, the identification of FHB resistance within breeding programmes is expensive and time consuming. The availability of molecular markers linked to the resistance loci should reduce the cost of producing FHB resistant varieties of wheat for cultivation in Europe.
We have full length clones of some wheat genes that may be associated with wheat tolerance to deoxynivalenol (DON) mycotoxin. They were isolated based on the fact that they were up regulated in progeny from a wheat cv. CM 82036 (DON tolerant) and Remus (DON intolerant) that inherited the DON tolerance of the former. These include genes of diverse function. These genes can be tested for their ability to confer DON tolerance upon plants.
Trichothecene hypersensitive yeast strains were developed which have reduced drug efflux due to inactivation of ABC transporters(pdr5,10,15), a detoxification gene (acetyltransferase ayt1) and a ribosomal protein affecting translational fidelity (rpsX). These strains require far less expensive toxin for full growth inhibition and are suitable hosts for identification of heterologous cDNAs that confer increased toxin resistance in yeast.
The encoded gene products could act directly or by connecting with yeast stress pathways. To distinguish whether or not this is the case several yeast genes with an important and redundant role in stress resistance have been inactivated: pdr1 pdr3 (pleiotropic drug resistance genes), yeast AP1 like genes (implicated in toxin and oxidative stress responses (yap1 yap2), and the transcription factors msn2 msn4 (stress response element binding.
We have generated about 10 million yeast transformants containing plasmids leading to the expression of wheat cDNAs under the strong PGK1 promoter.
The yeast transformants were washed off from nonselective transformation plates and stored at -70°.
Aliquots could be thawed and used to select for resistance conferring plasmids to any substance toxic to the yeast host strain (pdr5,10,15 ayt1, rps11). This should save time end expenses for potential patners needed to generate the library and transforming yeast.
We have gnerated a cDNA library from wheat, which was treated with deoxynivalenol. The primary library is a phage lambda library in vector lambda PG5 (Brunelli and Pall, 1993) with a complexity of 2.10E6pfu. From the amplified library we have generated plasmid DNA pools by Cre/lox mediated in vivo excision (more than 50% have inserts between 750 and 1500bp.
The library can be used as phage library for cDNA screening or the excised plasmid to transform yeast (the cDNA inserts are expressed under control of the PGK1 promoter).
A range of winter wheat varieties and lines, 111 in total, which were described as moderately to highly resistant (together with few susceptible checks) has been characterized for Fusarium head blight (FHB) in replicated experiments for 3 years.
The lines were tested using artificial inoculation with maize stubble distributed over the trial area in autumn, and by applying a spore suspension of Fusarium approximately three times during anthesis. Before and after inoculation with spore suspension the trial was kept moist by a mist irrigation system running for a few minutes 2-4 times a day during the inoculation period, around anthesis. For a period of about two weeks after anthesis, the mist irrigation was turned on in the morning and evening to extend the moist period during the night.
The evaluation for FHB was performed 2-3 times with a few days in between, from the beginning of symptom appearance until the more susceptible lines were fully diseases. The disease severity was assessed on a 0-9 scale were 0 is resistant and 9 susceptible, this scale corresponds to 0 to 100 % coverage of the ear with disease symptoms. Overall the inoculation system worked very reliably and good disease evaluation was made in two out of three years. One year disease failed to develop before natural senescence probably due to unusually cold conditions during anthesis.
As the tests were made in both research institutes, it is reasonable that they share the IP for the finding. 24-24 genotypes selected from the population CM 82036/Remus were used for the test having 2 QTLs 3B and 5A, 3B and 5 A separately and again a group without known QTLs. The 3B QTLs is effective against spreading, called also resistance component 2, 5A is effective against invasion, normally measured by the ratio of infected heads, called also resistance component 1.
These two QTLs are the two, which have the strongest effect on FHB resistance among QTLs identified until now. All other QTLs have medium or small effect. For this reason it was an important question, what is the function of these QTLs following field inoculation.
For this a two sites, two plot-replicated trial was used, where each plot replicate was treated by eight different Fusarium isolate. Following inoculation FHB rating on the field, FDK, toxin (DON) contamination, 1000 grain mass, seed No./head, test weight, yield response, DNA and ergosterol content was measured to see the scope of the QTLs. The most important lessen is that the group means for lines carrying 5A and 3B QTLs gave very similar performance with no or slight difference, even for toxin contamination.
The group having both QTLs gave significantly better effect, very high resistance as a synergetic effect of the two differing effect QTLs. This proved us, and all breeders that 3B is alone not effective enough to secure high level of resistance and the same is valid for the 5A QTL. For this reason an effective strategy is only that utilizes both QTL types in one plant. We think, this is the most important message of the test. Further, there was no difference for the different Fusarium spp. used. This is the first clear prove that individual QTLs are species non specific. This is again a highly important message for the breeders.
Additionally the QTL effect was strongest for FHB, FDK and DON (or for F. avenaceum moniliformin), the other traits were less influenced and difference between 3B+5A was not always different from the individual 3B and 5B effect. The last message was that the large effect QTLs were evenly identified in both institutes that worked with different methods in different environments. This means that these QTLs can be worked with under very different environments and conditions, which is again good news for the breeders.
We isolated RNA from deoxynivalenol-treated roots of wheat cultivar Frontana harvested 24 h post-treatment. This RNA was used to generate a cDNA library using the Clontech Smart cDNA cloning kit. This oduced from DON-treated roots (wheat cv. Frontana) harvested 24h post-incubation. This phage library was converted to a plasmid library (pTriplEx2). The library was then normalised in order to reduce the number of redundant cDNAs. The resulting plasmids were transformed into E. coli. Upon plating of the transformed bacteria, this resulted in excess of 3,000 clones, which were amplified and stored at 70oC.
We investigated the hypothesis that resistance to deoxynivalenol (DON) is a major resistance factor in the Fusarium head blight (FHB) resistance complex of wheat. Ninety-six double haploid (DH) lines from a cross between CM82036 and Remus were examined. CM82036 originates from a cross Sumai-3/Thornbird-6. The DH lines were tested for DON resistance after application of the toxin in the ear, and for resistances to initial infection and spread of FHB after artificial inoculation with Fusarium. Toxin application to flowering ears induced typical FHB symptoms. QTL analyses detected one locus with a major effect on DON resistance (LOD = 53.1, R2 = 92.6). The DON resistance phenotype was closely associated with an important FHB resistance QTL, Qfhs.ndsu-3BS, which was previously identified as governing resistance to spread of symptoms in the ear. Resistance to the toxin was correlated with resistance to spread of FHB (r = 0.74, P<0.001). In resistant wheat lines the applied toxin was converted to DON-3-O-glucoside as the detoxification product.
There was a close relation between the [DON-3-glucoside]/[DON] ratio and DON resistance in the toxin treated ears (R2 = 0.84). We conclude that resistance to DON is important in the FHB resistance complex and hypothesize that Qfhs.ndsu-3BS either encodes a DON-glucosyl-transferase or regulates the expression of such an enzyme. Investigations of a second wheat population, including most known wheat genotypes with a high resistance level to FHB, showed that the wheat lines Sumai3, Nobeokabozu, Wuhan and their derivatives expressed high resistance to DON. Also in these lines the applied toxin was converted to DON-3-O-glucoside. These data showed the latter mechanism of DON detoxification is a common mechanism present in all DON resistance wheat lines identified so far.
Results are published. The DON resistant lines described are commonly available for research and resistance breeding.
Total FHB resistance in wheat can be dissected into several resistance components. Resistances to initial infection (resistance component type I) and spread of FHB in the host (type II) are commonly accepted. Resistance component type III was described as insensitivity of wheat lines to the toxin, defined as the ability of the resistant cultivar to degrade DON. So far it was not clear whether DON resistance in wheat exists and whether it substantially contributes to FHB resistance in general. In this contribution we could not only show that DON resistance significantly increases FHB resistance, but we could also elucidate the biochemical mechanism of DON resistance.
For practical breeding these findings might have several consequences. We know that the DON-producing ability of the Fusarium isolates correlates well with their virulence. It is expected that the introduction of DON resistance to wheat will reduce pathogen virulence and will increase total FHB resistance, leading to less symptoms and lower DON contamination levels relative to DON susceptible wheat cultivars. The DON conjugate formed in DON resistant lines was indeed found in both artificially inoculated and naturally infected wheat samples. Yet, results obtained with a small set of wheat lines revealed that although resistant cultivars contained higher amounts of DON-glucoside, total content of DON + DON-glucoside is an order of magnitude larger in DON- and Fusarium-susceptible wheat cultivars. The biochemical fate of DON-3-O-glucoside in human and animal intestinal tracts is currently unclear. DON-3-O-glucoside produced in DON-resistant wheat lines could be a so called masked mycotoxin, which is not detectable with routine analyses but which might regain its biological activity after the glucose moiety is cleaved off in the intestinal tract. In this respect it is interesting to mention that the cultivar Sumai-3 , which contains the major QTL for FHB resistance Qfhs.ndsu-3BS, is the most widely used resistance source in the world.
A list of 111 winter wheat varieties and lines which were described as moderately to highly resistant (together with few susceptible checks) has been characterized for Fusarium head blight in replicated experiments for 3 years. The lines were tested using artificial inoculation with F. culmorum strains produced by P1 by spraying directly on the heads during anthesis. To insure enough humidity the application time was in the evening or just before proposed rainfall. Since our location is situated beside a river no further humidity control was necessary.
he inoculum concentration was 500.000 conidia/ml and application was done twice within one week for each plot. Evaluation of FHB was assessed in % diseased spikelets/ear( = severity) on a linear 0 - 100% scale 3- 4 times, beginning 10 days after inoculation. The scores from each year were calculated as AUDPC values (=area under disease progressive curve) to be able to compare years and locations. Highly significant differences between FHB resistance of the tested lines could be detected by variance analysis. Also a high accuracy and repeatability of our data can be seen, which has been calculated on basis of the intensive phenotyping by all involved project partners. A seed set of genotypes out of this screening nursery (excluding restricted breeding lines) is available for any interested breeding company. Resistance data of the whole set will be published by our consortium.
Hence any interested breeder has the possibility to compare and improve his artificial inoculation technique regarding the evaluated resistance level of the analysed genotypes from our consortium. The improved inoculation method is an efficient and reliable tool for us to select new genotypes within our wheat breeding program for FHB resistance to develop varieties with low mycotoxin content. Out of the most resistant lines from the nursery we already started a crossing program to develop more adapted resistance sources and finally new varieties.
Our goal was to compare, evaluate and improve artificial inoculation techniques of the participating partners (if necessary). The partners involved in this project have in part different inoculation techniques to investigate FHB resistance. Most of them use irrigation to apply moisture to improve infection. Inoculum is applied by spraying, or diseased plant residues are distributed on the soil. From the latter crop debris spores can infect the wheat ears. P1 and P5 apply the inoculum at flowering time of each individual genotype, whereas the other partners repeatedly spray all lines simultaneously during the flowering period of the lines.
Also the evaluation system was different. P1 and P5 evaluate each line at well defined time points after flowering, whereas the other partners evaluate all wheat lines at the same time independently from the exact flowering date. Also the assessment system (data scale) was different.
On top of that, it is well known that FHB resistance testing is influenced by environmental conditions. The resistance data of the different seasons assessed by each individual partner were in general highly correlated, indicating that the inoculation techniques result in similar or comparable resistance data over years (good repeatability).
In general the data of each partner were also highly correlated with the true resistance of the genotypes, indicating that each partner assesses the correct resistance level (good accuracy). Taken all these differences in inoculation methodology, assessment method and also locations and years into account, we were surprised that the resistance data of the different partners related pretty well. Interesting are the high correlation coefficients among the data from the breeders (P2, P3 and P4).
The data from the scientific partner P1 significantly correlate with the data from P2, P3 and P4. The data of P5 showed lower correlation with the resistance data of the breeders and only moderate correlations with P1. It is at present not clear what might be the reason for this. All breeders spray the inoculum several times during the flowering period, and assess the resistance of all lines at the same time point. This might be the reason why the data of the those partners correlate well with each other, but less with the data of Partner 1 and 5.
The latter partners inoculate and evaluate each individual wheat line at precise time points during and after flowering. Late flowering and therefore late inoculated wheat lines might show less symptoms as compared to the early wheat lines, simply because the period of time for disease development is shorter for the late genotypes. Partner 1 uses extensive mist irrigation to improve infection success. The late genotypes are irrigated during a shorter time period after inoculation as compared with the early genotypes.
This might result in a stronger disease level on the early genotypes. P5 uses bags to cover the inoculated ears with after inoculation. If inoculation is done during the morning temperature might rise in the bags on sunny days. This factor might influence infection if temperature in the bags would be to high due to strong insolation. These considerations should illustrate that there exist further possibilities to improve our inoculation methodology. The investigated wheat lines could be grouped according to flowering time in order to reduce mist irrigation to a minimum.
A small set of wheat lines could be incorporated as standards or controls to compare infection success of different inoculation days: especially cold temperatures reduce infection success, and temperature between different inoculation days might vary significantly. Other methods of inoculum application such as the distribution of Fusarium infected plant debris should be considered.
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