Reporting
Period: September 1, 2004 to February 28, 2005
1.
Research
Objectives:
Fusarium fungi (F. graminearum, F. culmorum, F. avenaceum, F. poae, F. tricinctum, F. equiseti, F. verticillioides, F. oxysporum, F. subglutinans, F. sporotrichioides and Microdochium nivale) are an important problem in the whole cereal food and feed chain because they contaminate the grain with mycotoxins (trichothecenes, [e.g. deoxynivalenol (DON), nivalenol, T2-toxin], which cause serious illness and immuno-repression in humans and animals. The objective of this project is to reduce mycotoxin contamination in Europe’s most important crop wheat at the start of the production chain by means of improved Fusarium Head Blight (FHB) resistance. The development of new cultivars with low risk of mycotoxin accumulation is therefore of utmost importance. To meet the goals a multi-disciplinary approach involving plant pathology, plant breeding, molecular genetics using phenotyping, genomics and in vitro research methods is applied. The project has eight partners cooperating in 3 work packages:
To improve the efficiency of conventional selection for resistant lines, artificial infection in mist irrigated fields and under controlled conditions are performed [(Workpackage 1 (WP1)].
This will enable the team of participants to develop molecular markers for selection of FHB resistance at the genetic level by QTL mapping of segregating populations as well as high resolution mapping and selective genotyping of resistant germplasm (WP 2). We already have three mapping populations and two more mapping populations are under development.
The search for innovative selection techniques (in vitro selection, candidate resistance genes and pathogenesis related protein) for resistance against the fungus and toxin accumulation are part of further trials of the project (WP 3). Please note, that based on recent scientific developments some required adjustments of the original technical annex were approved by EU.
Results and
Milestones:
The accessions in nurseries containing advanced germplasm and mapping populations were planted during Autumn 2004 and spring 2005 and will be evaluated for their FHB resistance during Summer 2005. According to the milestones and deliverables due after forty-two months, experiments under field and controlled conditions as well as laboratory research were completed and major results are given below.
Subtask 1:
Phenotyping of a wheat screening nursery
Resistance data are now available over 2 seasons. FHB data over at least
two seasons/locations are required due environmental factors influencing the
resistance data. Severity was determined by all partners in this subtask and
severity 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) and
Type II resistance (resistance against colonization). Separate resistance
components (Type I and II) were investigated by P1 only. Incidence primarily
reflects Type I resistance, whereas both spread and wilting measure Type II
resistance. Type I and Type II resistance seems not to be coupled. For example
the genotype Frontana has an excellent overall resistance based on AUDPC of the
severity. According to the data for spread and wilting, this genotype has a low
Type II resistance. Hence, the good FHB resistance of Frontana is due to a high
Type I resistance. Such data are interesting, because combining a high Type I
and Type II resistance could further improve FHB resistance.
The second goal of this Subtask was to compare and (if necessary) improve inoculation methods of the participating partners. P1 and P5 use elaborate inoculation techniques, the main difference between both partners is the method used to ascertain humidity during inoculation: P1 uses a mist irrigation system, P5 uses bags to cover the treated ears. The participating breeders use different methods ranging from spraying the inoculum on the ears up to combinations of sources of inoculum on the soil surface and spray application, but no bags. Based on the data of two years now, a correlation analysis was done with the severity resistance data of each partner for each year separately. 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 show no correlation with the resistance data of the breeders and only moderate correlations with P1. These findings are repeated over two seasons now. Based on these data over 2 years, the inoculation and evaluation methods of P1, P2, P3 and P4 seem to produce comparable results. It seems than an additional analysis of the data will be necessary according to inoculation time and ripening groups to find explanation(s) for the often different behavior of the materials in different locations.
Subtask
2:
Phenotyping of mapping populations
The CM 82036/Remus population could be tested successfully. All parameters that were planned were evaluated. This will be valid also for the other MPs. Next year the Frontana/Remus population will be tested again. All data speak for the fact that 5A chromosome is at least as strong at spraying inoculation than 3B, in some cases better, even the difference is not always significant. These differences could be shown first of all at the stronger aggressive isolates, at the less aggressive isolates QTL group differences are often not significant. The phenotyping results at MP1 show clearly that the different traits are differently influenced by the QTLs. The clearest picture was seen at AUDPC, FDK ergosterol and DON values, the other parameters were less differing. The closest correlations and largest MS values were counted for these parameters, the others less informative. This conclusion was supported by the second year’s results, too. Even so, the FDK and FHB values often disagreed; this means that the two parameters cannot replace each other. It has several reasons. The evaluation of the FHB symptoms finishes 26-30 days after inoculation, but until harvest a month additional time is minimally recorded. In this period the fungus spreading continues and rains can enhance this process.
Subtask 3:
Phenotyping of high
resolution mapping populations
Resistance
evaluation of both fine mapping populations (for 5A and 3B) proceeded well. Lack
of seeds forced us to test the 5A population on one location only. For the next
season (2005) the tests is done on two locations (P1 and P5). Highly significant
differences among the lines were detected for both populations. In order to have
reliable results for FHB resistance we need at least data over two seasons.
Hence the results will be discussed in detail in the report of next year.
Subtask 4:
Measurement of type III (trichothecene) resistance
It was shown already
in mutants of Gibberella zeae that deoxynivalenol production is an
important virulence factor of the fungus. So far it was not proven that
resistance towards deoxynivalenol is an important factor in overall FHB
resistance. In this subtask we were able to provide evidence that after
application of DON in the ear, the Qfhs.ndsu-3BS improved DONR
dramatically (AUDPC was reduced from 12.47 to 2.37 units). This QTL is also a
major QTL improving FHB resistance towards the fungus. Moreover, QTL analyses
showed that Qfhs.ndsu-3BS is the only gene coding for this trait in the
CM*Remus population (LOD = 47, R2 = 0.90). Interestingly we could
mimic with DON most of the symptoms which are otherwise caused by the fungus,
including bleaching of the spikelets, spread of the symptoms both in acropetal
and basolateral direction and wilting of the complete ear above point of toxin
treatment. Qfhs.ndsu-3BS is only playing a role in Type II resistance
(resistance towards spread of the fungus in the ear after successful infection).
The DON resistance data only correlated with Type II resistance (r = 0.78) and
not with Type I resistance. This indicates that 1) DON resistance is an
important factor in the complex FHB resistance, and 2) DON resistance is
probably part of Type II resistance and not a separate resistance component
(Type III). We consider this finding important, but it does not explain all
differences we mentioned. Other processes and gene effects influencing DON
accumulation can also be present. This is obvious when we look genotypes without
type II resistance and have low DON in spite of higher FHB severity.
So further research in this field is important and we are now in the
position to put better questions in future experiments. In the screening nursery
we could identify additional wheat lines with excellent DON resistance such as
Sumai3, Nobeoka Bozu, Triticum macha etc. From other experiments we also
have an idea of the possible resistance mechanism on a biological/physiological
level: the DON is conjugated to glucose. If we test DON resistance using leaf
disks or seeds, we were able to find highly significant differences among the
wheat lines tested, but no significant correlations with FHB resistance. Hence,
we conclude that in these tissues other DON resistance mechanism are working
which are probably not related with DON resistance acting in the ears.
Subtask 1:
QTL mapping in segregating populations
Population 1 and 2 were
completed earlier, work focussed therefore on population 3.
P4: During September 2004 P8
delivered additionally (new) DH lines to improve the map in several locations.
This WP is also over-fulfilled typing additional 24 DH lines with several SSRs
to refine the map of P8. The files with raw data have been forwarded to P8 in
Dec.2004 (see details in report of P8).
P8: Analysis to date has indicated the presence of both stable and unstable QTL for FHB resistance. In light of this, the whole population was re-tested for FHB resistance by spray inoculation in a poly-tunnel at JIC. Disease progress was assessed at several time points following inoculation to produce an “area under the disease progress curve” (AUDPC) assessment. QTL for FHB resistance were detected on chromosomes 4D and 6B in similar positions to those found previously. Thus these two QTL for FHB resistance appear stable across years at this site. In addition, the population was screened for FHB resistance in the field by partners 1 and 5. Seed was supplied to both partners and plots were grown and inoculated at anthesis by spray inoculation. Results from partner 1 have been analysed and a QTL on 4D, similar to that observed in polytunnel tests was observed. This QTL appears stable across years and environments. The reason for the absence of detection of the 6B QTL is not known. Additional QTL were also detected within the field experiment but these have never been observed in experiments within polytunnels. QTL analysis of data from field trials of partner 5 has also been analysed during this reporting period. A QTL for visual disease was observed on chromosome 4A. Three QTL were detected for ‘Fusarium damaged kernels’, these were on 4D, 5A and a linkage group believed to be part of 1B. In addition a QTL for yield loss was detected on the same 1B-related linkage group. Overall, the 4D QTL has been shown to be stable over all environments with other QTL also evident in some instances. For example QTL on 1B have been observed in both polytunnel and field environments (it is unclear whether these represent single or multiple QTL on the same linkage group. A QTL on 4A has been active in field trials of both partner 1 and 5.
Subtask 2:
High resolution mapping of FEB resistance
Phenotyping:
The selected recombinant lines of the high resolution mapping populations were planted in replicated field experiments again in spring 2005. We have planted two replications of the 3BS fine mapping population and 2-4 replications of the 5A population in Tulln. In addition both populations have been planted in Szeged (Hungary) for replicated phenotyping.
Genotyping:
We have attempted to continue and refine marker analysis with a focus on the 5A fine mapping population with additional SSR markers that were found to map in the target interval in other reference maps (e.g. graingenes database). The SSR markers Gwm443, Barc135, Barc316 and Barc122 were chosen for further genotyping of the 5A population. Gwm443 and Barc135 were polymorphic between CM-82036 and Remus, while Barc122 and Barc316 were monomorphic. We analysed the 5A high resolution population therefore with Gwm443 and Barc 135. Both markers were monomorphic on this population of recombinant inbred lines, all lines had the ‘Remus’ allele. This can be explained by the fact that the DH-Line which was originally used for creating the high resolution mapping population (D2-54_T) was already fixed at these two loci for the Remus alleles, and these markers did therefore not segregate in the population. An unexpected finding was, that the 5A high resolution mapping population showed extremely suppressed recombination in the target interval, where we expect the QTL on 5A to reside (Qfhs.ifa-5A). It is unclear so far, whether this is a general phenomenon happening in ‘all’ wheat crosses or whether this fact is specific to the cross we have been working on, which was derived from D2-54_T (resistant) x Remus (susceptible). In order to answer this crucial question, we have started to analyse four additional crosses with linked SSR markers mapping in this region in detail. We therefore generated new segregating populations for the 5A QTL. We used crosses of lines possessing the 5A QTL (resistant lines derived from CM-82036, which contribute the 5A QTL) with susceptible lines that are NOT related to the susceptible parent Remus. In four populations of 120 lines each we extracted DNA from F2 plants using similar protocols as in the previous years for genomic DNA extraction. We started to genotype these lines with SSR markers that were closely or completely linked in the original mapping population 1 and the high resolution mapping population. This work is in progress and we expect the results until month 48.
Candidate gene markers:
A SNP assay was designed by P7 for the RPL3 (ribosomal protein L3) gene locus Ta_RPL3_A3. We have mapped this gene on chromosome 5A in the Qfhs.ifa-5A region in our mapping population 1 (see previous reports). In order to map this gene more precisely, we also mapped the same gene in our high resolution mapping population for 5A. We have sent DNA of the 5A high resolution mapping population and of Chinese Spring deletion lines to P7 and P7 has analysed these lines with their SNP assay and reported data back to P1. P1 has analysed the data using the software Mapmaker and could confirm that the gene maps indeed in the Qfhs.ifa-5A region. The map position of Ta_RPL3_A3 is within the region of suppressed recombination mentioned above. Based on the analysis of Chinese Spring deletion lines, the map position of the gene was confirmed on the short arm of chromosome 5A.
During the first year a set of 100 SSRs especially suitable for phylogeny analyses had been selected, evaluated and established: According to adjustments to the screening nursery together with P1, finally 96 lines had been chosen for genotyping. DNA had been extracted, quality of DNA checked and typing with 64 SSRs had been achieved during year2. The raw data, additional cluster trees and the similarity scores had been added as an electronic supplement to the second year report. 96 SSRs including SSRs from regions where QTLs are detected (ongoing work at P1 and P8) have been investigated on the screening nursery up to date resulting in 111 loci (647 alleles). This work is finished. The current version of dendrogram and similarity matrix has remained the same as in the last report.
Subtask 1: A novel in vitro strategy for early screening of FHB resistance in adult cereal plants
We have determined that wounded leaves of cv. Remus are much more susceptible to DON than those of cv. CM 82036. We will now test how this DON sensitivity is inherited in the CM 82036 x Remus double haploid population.
Subtask 2.1: Candidate genes for resistance, toxification, detoxification and toxin efflux
Ribosomal protein
P8
found that few sequence differences are available that permit the design of PCR
primers to specifically amplify this gene. The phase 1 PCR protocol for specific
amplification of the complete RLP3 sequence from 4B has been refined.
DNA samples (provided by P1) from a high resolution mapping population with recombination events between markers flanking the 3A QTL were analysed using the SNP marker for RPL3-A3 by P7. Furthermore, wheat lines with chromosomal deletions in the QTL region were characterized by P7. The genotyping results were transferred to P1. The results indicated that also in the high resolution population the marker for RPL3-A3 is within the Fusarium resistance QTL interval, we therefore cannot exclude that RPL3-A3 is involved in Fusarium resistance.
New
goal: Characterisation of the effect of RPS11 dosage on DON resistance
To test whether in the yeast strains having RPS11 under a regulated promoter really have an altered stoichiometry of ribosomal proteins, we have made constructs allowing addition of a C-terminal epitope-tag to the chromosomal RPL3 gene. Candidate transformants having tagged both RPL3 and RPS11 as sole source of the respective ribosomal proteins were obtained. Western blotting experiments with such strains should allow to get clear evidence whether changes in DON resistance are correlated with changes in relative levels of the ribosomal proteins.
Subtask 2.2:
ATP binding cassette
(ABC) transporter proteins
Extensive experimentation has been performed to determine the most appropriate methods for real-time RT-PCR assays. The most critical aspect of experiments is the appropriate analysis of data. A wide number of methods have been employed and all have been shown to be flawed in some respect. Robust analytical methods have been developed and adopted for studies of expression of PDR-like sequences.
Expression of the DON-responsive PDR5-like sequences (A1, A2, A3, C1, C2, C3) have been examined in root and shoot tissues following treatment with various compounds in a series of replicated experiments to confirm the effects. Treatment compounds included DON, NIV, abscisic acid, cycloheximide, jasmonic acid, and salicylic acid.
In contrast to the A class, treatment of roots with compounds did not reduce expression for all three C types. DON and cycloheximide enhanced expression of C1 and C2 in shoots of root-treated seedlings relative to controls. No stimulation of C3 was observed for any compound but abscisic acid, jasmonic acid, and salicylic acid significantly reduced expression.
DNA clones of PDR5-like sequences
from wheat
A cDNA obtained from the DON-responsive
PDR5-like sequence C3 was used to probe a wheat BAC library of the variety
Chinese Spring. A large number of clones were identified in the library using
this probe. PCR primers specific to C3 were used to amplify from hybridising
clones. Amplification products were sequenced and compared to the original cDNA
DON-responsive sequences obtained earlier. Six sequence haplotypes were observed
and PCR primers designed to putative intron sequences for use on DNA. PCR was
performed on nullitetra lines to determine the chromosomal location of the
clones. ST 2.3:
As previously described we have the evidence that GST mediated detoxification of DON exists:
Subtask 2.3:
Glutathione S-transferase (GST) homologs as candidate genes involved in
trichothecene detoxification
Significant progress has been made in collaboration with chemists (synergy with Austria genome project GEN-AU FUSRIUM.) Based on tests with various trichothecenes and data of LC-MS/MS measurments, we have a putative structure for the glutathine conjugate of DON formed by yeast. It is at present unclear whether other transporters can also take over the function in the presence of the high amounts of glutathione added. Since the toxicity of DON is reduced about twofold, but the concentration of free DON in the medium remains more or less unchanged, we do not think that glutathione reacts directly with DON in the medium. Further studies are necessary to support or falsify the hypothesis that DON-glutathione adduct formation is an intracellular enzyme-dependent reaction.
Subtask
2.3B: New task
(with prior EU approval): Screening for wheat genes conferring trichothecene
tolerance using a yeast heterologous expression system
P7 developed a DON sensitive yeast strain, and during this project, and with EU approval, it was decided to screen a cDNA library from DON-treated wheat tissue in this yeast system to determine if any transcript conferred DON tolerance to the yeast. The objective of this subtask was therefore to obtain and characterise wheat transcripts that conferred DON tolerance to yeast.
The screen for wheat cDNAs which confer DON resistance when expressed in yeast has revealed 8 different types of insert sequences so far. Several of the genes were found more than once (either as cDNAs of different length or also as clones originating from homeologous loci). To learn more about the possible mechanisms of resistance the DON resistant candidates were tested for cycloheximide resistance, and resistance against canavanine, paromomycin, hygromycin, cadmium and ketokonazol). With a new screening method we hope to increase the chances of finding the desired wheat glucosyltransferase inactivating DON.
Subtask
2.4:
Identifying novel candidate genes for trichothecene tolerance/degradation:
Relationship between QTL3A and
candidate genes for trichothecene tolerance from the differential display study
From our differential display work we had genes and retrotransposons that were more DON responsive in roots of cv. CM 82036 than in those of cv. Remus. We tested the relationship between the expression of these transcripts in roots, coleoptiles and heads in response to DON, in the CM 82036 x Remus population. The expression of no gene was exclusively correlated with inheritence of the FHB QTL (QTL 3A) associated with DON tolerance. Genes encoding phenyl ammonium lyase (PAL) and a glucanase showed elevated expression in heads in response to DON in lines carrying QTL 3A + 5B, QTL 3A and in lines carrying QTL 5A, but to a lesser extent in the latter group.
Comparison of gene sequence in
different cultivars.
For four genes, no sequence differences
were detected that would affect amino acid sequence. Rather than sequence more
genes from the differential display experiment we are now focussing our efforts
on the DON-responsive genes from the microarray experiment as (a) we have clones
of these genes and (b) the design of the microarray experiment means that any
candidate genes are more likely to be associated with DON resistance inherent in
QTL 3A.
Candidate genes for trichothecene tolerance from microarray analysis
Preliminary results indicated that, in response to DON, twenty two genes are upregulated in lines carrying QTL3A and in CM 82036 that are down-regulated in Remus and other double haploid lines that do not carry QTL3A. A further 120 genes are significantly more up-regulated in lines carrying QTL3A and in CM 82036 than they are in Remus. These genes are currently being sequenced.
CM 82036 x Remus population Fusarium
head blight experiment.
We have conducted a FHB experiment of the CM 82036 x Remus double haploid population for analysis of the response of DON-responsive genes to F. graminearum so that we can study the relationship between phenotypic FHB data and gene expression data. Heads were inoculated with the pathogen at mid-anthesis and harvested at 4, 24 and 72 h post-inoculation for subsequent RNA extraction and gene expression studies.
Subtask
3:
Candidate genes for inhibition of toxin accumulation and fungal growth
Subtask
3.1:
Inhibition of toxin accumulation (completed after 24 months)
Subtask
3.1B:
New task Characterization of UDP-Glycosyltransferases (UGT) (with prior EU
approval)
In the light of current results, a more focused homology search is now possible. Yet, the approach to identify the most similar wheat homologs and to test the cDNAs by expression in yeast seems risky and is pursued with low intensity. We hope that the functional approach – making a cDNA library and selecting resistant clones in yeast – will yield the right gene.
Subtask
3.2: Antifungal
proteins (Completed
after 24 months)
Subtask
3.3:
Antifungal compounds and inhibition of fungal toxin production: markers for
candidate benzoxazinoid sunthesis genes of wheat (Terminated
after 24 months_
Future Actions:
At the present stage, future actions are not required. All actions envisaged in the workplan can be fulfilled in due time. The few deliverables or milestones, which have been marked as “delayed” in the 42nd month report, can be achieved by being addressed until the 48th project month.
2. Exploitation and Dissemination
1. The Fucomyr project was presented at the Mycotoxin Cluster Dissemination Day in Brussels in October 2004 by Marc Lemmens (Talk), at the BCPC meeting in Glasgow and at the 2nd International Symposium on Fusarium Head Blight incorporating the 8th European Fusarium Seminar in Orlando, FL, USA in December 2004. A large number of results generated by the project was presented at the 2nd International Symposium on Fusarium Head Blight incorporating the 8th European Fusarium http://www.scabusa.org/forum.html#isfhb2
The report of individual members of the consortium can be viewed here: Participation SCAB Forum
2. The Fucomyr project website is active and updated regularly on the Boku-server (http://www.boku.ac.at/fucomyr/). The website covers numerous aspects of the project like the description of workpackages, meetings, publications, public awareness issues and provides links to other Fusarium and Mycotoxin related websites in Europe and beyond. A password protected part of the website was established and can only be accessed by the project partner, which utilize this area for project progress monitoring and project interaction activities.
3. Poster and leaflets: To report progress of Fucomyr to the scientific community and plant breeding industry during conferences and fairs, the poster was prepared. The current leaflet is a reduced image of the poster. Both, poster and leaflet is updated on a yearly basis, displaying the future progress of Fucomyr. The poster is effectively positioned at Breeders Days, Schooldays, open Days, Science Weeks and Agricultural fairs within all six European countries of the project partners. The poster is also available in Danish, German and Hungarian.
4. Publications: Several manuscript are accepted for publication, by now in the peer-review process or in its preparation stage. Besides this, new results are communicated by poster and oral presentation and by responding abstracts in conference proceedings. We always acknowledge the financial support by the European Commission.
3. Other reports:
Project management and coordination
Ethical aspects and safety provisions