Improving Fusarium Head Blight Resistance in Barley | ARDI | Agri-Food Research & Development Initiative | Manitoba Agriculture, Food and Rural Initiatives

Fusarium head blight (FHB), incited mainly by Fusarium graminearum, has recently emerged as the most serious disease of barley in the eastern prairie region of Manitoba and Saskatchewan, causing millions of dollars in damage. Although it can reduce yield, the most significant effect is in reducing quality due to the accumulation of deoxynivalenol (DON) mycotoxin in the seed. To be accepted for malting and brewing purposes, DON must be non-detectable (i.e. less than 0.5 ppm) in barley. As a result of the recent FHB epidemics, producers have been unable to take advantage of the lucrative 6-row white-aleurone malting barley market in the United States. DON is also a problem in the livestock industry, particularly to hogs since they are very sensitive to this mycotoxin. Feed barley has been downgraded or even rejected because of DON content, and uncontaminated barley brought into Manitoba for the hog industry at considerable expense. Most barley varieties currently grown in the eastern prairie region are highly susceptible, particularly the 6-row white-aleurone varieties preferred in the United States. Some barley varieties, particularly the two-row malting varieties such as AC Oxbow, AC Metcalfe, CDC Stratus and CDC Kendall, appear to be more resistant, but will still accumulate DON at high enough levels to be rejected for malting purposes under moderate to heavy epidemics of FHB. Our knowledge of the FHB reaction of all current barley varieties is incomplete and limited for newly registered varieties. Such knowledge would assist in making recommendations to producers on variety selection and in deciding which varieties to use for crossing in barley breeding programs. Evaluating the FHB resistance of entries in barley registration tests and advanced breeding lines from breeding programs would provide this information even sooner, allowing barley breeders and producers to make better choices. Some barley accessions from other areas of the world are more resistant to FHB, but are very poor agronomically and susceptible to other diseases, such as stem rust, net blotch and spot blotch. Incorporation of this “exotic” FHB resistance into locally adapted varieties would hasten the development of FHB resistant varieties and reduce the major losses caused by this disease. Improving genetic resistance to FHB would improve the quality of barley and allow producers to regain access to the export market for malting barley, particularly in the United States. It would also allow producers to grow feed barley with low DON levels to meet the needs of the expanding hog industry. The feeding industry would be assured of a good supply of locally grown, uncontaminated barley at lower cost since freight would be reduced. Improving FHB resistance would also reduce the need to use costly fungicides which are not 100% effective in controlling the disease.

The utilization of plant tissue culture has the potential to allow rapid evaluation and selection for FHB resistance and rapid development of new varieties through in-vitro screening and doubled haploid (DH) technology. This latter allows the production of homozygous lines in one generation by regenerating fertile plants from immature pollen (or microspores) in the laboratory, which reduces the time scale of varietal development by about two years. It could also result in the recovery of genetic material with enhanced tolerance to FHB infection and mycotoxin accumulation. Fusarium-produced mycotoxins, including DON, are thought to play a role in the infection process, and hence could be used for selecting tolerant germplasm in tissue culture. In-vitro selection has been successfully applied to several plant-pathogen systems, including FHB of wheat and triticale, but its application has not been investigated in barley. The anther culture DH system implemented at Agriculture and Agri-Food Canada Brandon was used to investigate this avenue with the aim of rapidly improving FHB resistance in all classes of barley.

The overall goal of the project was to improve FHB resistance of barley. The key was to establish a large nursery in which thousands of barley lines from Agriculture and Agri-Food Canada Brandon and Crop Development Centre/ University of Saskatchewan breeding programs can be evaluated for their reactions to FHB and tested for DON content. Such a nursery was established at Agriculture and Agri-Food Canada Brandon, with the Cereal Research Centre providing additional information and back-up for high priority material in a smaller nursery already established at Glenlea. Dr. Savard of Eastern Cereal and Oilseed Research Centre, Ottawa, provided the DON testing critical to the project (maximum of 5,000 DON determinations per year).

Work was done on all of the above objectives simultaneously each year. ARDI funds were used specifically to match the Western Grains Research Foundation grant for objective 5) on in vitro selection and to support evaluation of the Manitoba (Agriculture and Agri-Food Canada Brandon) germplasm as outlined in objectives 1) and 4).

Procedure and Project Activities:

The project was initiated in 2000 with the establishment of a large nursery at Brandon, Manitoba, in which thousands of entries could be evaluated for FHB resistance. Over the three years, the total number of entries included in the nursery, and the methods by which they were screened differed to some degree. In 2000, the nursery included 12,416 plots consisting of 7,680 1.5-m rows, 1,536 0.9-m rows and 3,200 hill plots. Of this total, 3,406 plots were harvested from the nursery. Significant variation associated with FHB disease reaction has been observed in barley. Evaluation of entries using hill plots was less consistent than rows, thus they were discontinued. In 2001, the nursery grew to 16,200 plots of 1,800 1.5-m rows and 14,400 0.9-m rows, with higher priority materials grown in the longer row type. A total of 7,882 rows were harvested from the 2001 nursery. The 0.9-m rows demonstrated a comparable level of variability with the 1.5-m rows, and thus were more efficient for assessing FHB reactions. In 2002, the entire nursery of 14,400 plots was sown to 0.9-m rows, which was less than the total number of rows sown in the previous year due primarily to seed shortages at the Crop Development Centre / University of Saskatchewan because of drought conditions in 2001. However, the number of rows harvested in 2002 actually increased to 9,728 due to increased replication of entries and use of composite samples for DON testing.

The nursery was inoculated with grain spawn (i.e. corn seed infected with 3 isolates of Fusarium graminearum) spread on the ground 3-5 times at weekly intervals starting before the earliest lines in the nursery headed, and irrigated to promote fungal development. In 2001, a supplemental experiment was conducted to assess the use of Tilt and Dithane fungicides to control Cochliobolus sativus (causal agent of black point, kernel discolouration, spot blotch, seedling blight and common root rot in barley). C. sativus may confound FHB research both through incorrect diagnosis of disease symptoms and inter-species competition. Although the experimental results were inconclusive, they suggested that Tilt could be used without adversely affecting Fusarium development. In 2001 and thereafter, 1-2 blanket treatments of Tilt were applied to the nursery prior to the first application of corn inoculum. All entries were rated visually on a 0-5 scale (0 = no symptoms, 5 = severe symptoms) about 3-3 ½ weeks after heading. A lodging score was also assigned at that time using a 1-9 scale (1 = erect, 9 = flat). Depending on the purpose of a test, the visual FHB scores were often used to select genotypes with the most promising FHB resistance for harvest and DON analysis.

Of utmost importance to the malting barley and animal feeding industries is the production of a FHB resistant variety with low DON content. In barley, visual FHB scores have largely shown low to moderate correlation with the level of toxin in the grain so that when conducting FHB research on this crop extensive DON testing is required. Grain harvested from a FHB nursery plot was cleaned, and a 20 g subsample was ground with 1 gram being sent to Dr. Marc Savard’s lab at Eastern Cereal and Oilseed Research Centre, Ottawa, for DON analysis using the ELISA technique. We submitted 2,770 samples for DON analysis in 2000, 6,198 samples in 2001, and 5,204 samples in 2002. Along with 50 additional DON assays conducted on samples originating from a 1999 FHB nursery, several hundred samples from the current study and their corresponding DON data (determined by ELISA technique) were used by researchers at the Crop Development Centre, Saskatoon, to develop a calibration for NIR to predict DON levels in barley.

To monitor environmental fluctuations over the nursery, repeated check sets were positioned after every 50 plots of experimental lines. It has been clear from the variation observed among checks that the expression of this disease has a strong environmental component. Expression of DON content was strongly influenced by year; DON content exhibited a ten-fold increase in 2001 over 2000, with 2002 levels falling somewhere in between. The effect of row type has also shown strong interaction with year. The improved FHB resistance of 2-row types seen in previous years was less apparent in 2002, which may have resulted from a prolonged dry spell at the beginning of the season favouring the earlier heading 6-row types. Interpretation of DON data has not been straightforward, and has required multiple years of testing for reliable conclusions.

Over the last 3 years, we have used a FHB nursery in Hangzhou, China, to provide a second cycle of FHB screening each year over the winter months and an assessment of materials in a FHB nursery free of C. sativus. In 2000, we sent 1,000 entries, which was subsequently doubled in 2001 and 2002. Our Chinese colleagues determined days to heading, % diseased spikes, % infected seeds and a disease index. Overall correlations between the data from China and Brandon have generally been low, although similar patterns were observed among genotypes.

Specifically supported by ARDI, in vitro selection (IVS) in conjunction with DH production using anther culture was evaluated as a tool for generating or enhancing new and existing sources of FHB resistance and for screening segregating populations for FHB resistance. In vitro selection was applied to three types of parental materials within these studies: current varieties (two- and six-row, covered and hulless), FHB resistance sources, and populations segregating for FHB resistance. Several methods have been applied to produce the different IVS groups, including variation in the types of mycotoxins applied to the selective growth media, the concentrations at which they were applied and the timing. In addition to DON, several other mycotoxins produced by Fusarium spp., such as 3-acetyl deoxynivalenol (ADON) and T2, were employed in some experiments. Cells with tolerance to the mycotoxins were regenerated into plants. Since mycotoxins are involved in the infection process, it was hoped that tolerance to these in tissue culture would be translated into improved FHB resistance in the field. DON content of IVS lines was compared in FHB nurseries to the parental genotypes or the control DH lines produced in the absence of mycotoxin in the anther culture system.

No useful data were obtained from the FHB nursery at Glenlea over the three years due to unfavourable environmental conditions there. This nursery was intended as a backup for the larger nursery at Brandon, and constitutes a replication of higher priority materials. However, Dr. A. Tekauz’s crew from the Cereal Research Centre has provided critical help in rating the Brandon nursery.

In addition to ARDI, this project also received financial support from the Western Grains Research Foundation Barley Check-Off and Endowment Funds, Saskatchewan’s Agriculture Development Fund, and Agriculture and Agri-Food Canada’s Matching Investment Initiative program.

Results and Discussion:

Although improving FHB resistance will be a long-term effort, good progress was made during the three years of the project. Valuable information was provided to producers through the Manitoba seed guides (and equivalent in other provinces) on the FHB resistance of current barley varieties, allowing producers to make informed decisions. Without this project, the information would have been fragmented and incomplete. It also gave barley breeders a better idea of the FHB resistance of their varieties so that they can plan their breeding efforts accordingly. One of the disappointing findings of the project was that the FHB resistance of some two-row malting varieties, like AC Metcalfe and CDC Stratus, did not appear to be as good as we had thought at the outset of the project. Perhaps the resistance of these varieties breaks down more under the heavier Fusarium graminearum infection levels observed in 2001 and 2002. A number of two-row varieties appear more resistant to FHB than AC Metcalfe but they tend to be early, highly susceptible to spot blotch or do not perform well under Manitoba conditions. Few six-row varieties surpassed CDC Sisler, although the six-row blue-aleurone malting varieties, Argyle and Tankard, appeared to be more resistant. However, there is no longer any market for this class of barley. Bedford and Bronco were best six-row feed varieties and they were comparable to CDC Sisler in DON content. Two-row hulless barley varieties, like CDC Freedom, look very promising and may have the lowest DON content of all classes of barley, but unfortunately this class has been struggling for a market share in recent years. The six-row hulless varieties, AC Hawkeye and AC Bacon, compare favourably with two-row varieties in terms of DON content. The hulless trait may be partly responsible for this, but in looking at the six-row group in particular there are some hulless varieties that have very high levels of DON.

The project also provided data on FHB resistance for potential varieties in the western Canadian cooperative registration trials. Without this project, there would have been no data on this material at all since the FHB nursery for barley at Glenlea failed all three years. Not many new varieties were registered over the past 3 years with improved FHB resistance over AC Metcalfe (two-row check) or CDC Sisler (six-row check). The two-malting varieties, Calder and CDC Goodale, may have lower DON content than AC Metcalfe, while the two-row feed variety Ponoka from Alberta also looks promising. One of the most promising lines in the two-row cooperative test during this time was TR361 which was supported but not registered due to high susceptibility to spot blotch, an important disease in Manitoba. From the hulless cooperative test, HB364 from the Crop Development Centre was probably the most promising in terms of FHB resistance to be registered. It is expected that more lines with improved FHB resistance will be entering the registration tests over the next few years as a result of the research done under this project.

Germplasm exchange has been occurring with researchers in eastern Canada, United States and ICARDA/ CIMMYT. Since these researchers have been working at this problem longer than we have in western Canada, we hope to identify FHB resistant lines that we can use for crossing purposes to improve FHB resistance. In 2002, we participated in the North American Barley Scab Evaluation Nursery with American researchers for the first time. Although we only had 8 entries in the test, our lines performed reasonably well and were comparable to American lines specifically selected for FHB resistance. These elite lines may have the potential to become new varieties in western Canada or serve as parents for crosses. It is hoped that germplasm exchange and collaboration will build on this base and increase over the years ahead.

The best known sources of FHB resistance are CI4196 in two-row and Chevron in six-row types. Both are undesirable from an agronomic point of view, susceptible to other diseases and have unacceptable quality. Also, their FHB resistance will breakdown if infection levels are high enough. It would therefore be desirable to identify new sources of resistance to complement them or that are in a more desirable background. For this purpose, we have screened a large number of lines from Plant Gene Resources of Canada, as well as advanced breeding lines from European and Australian barley breeding programs. A number of lines have been identified with promising FHB resistance, but additional testing will be needed to confirm the results and determine their suitability in terms of agronomic, disease resistance and quality traits. This is expected to be a long-term process.

All barley breeding programs in western Canada have at least some lines with moderate resistance to FHB surpassing AC Metcalfe or CDC Sisler. Most of the moderately resistant lines in the registration trials to date were identified in existing breeding material, but new lines specifically bred for FHB resistance are now being advanced through the breeding programs. Of particular note are 25 Agriculture and Agri-Food Canada Brandon breeding lines from 10 crosses involving known sources of “exotic” FHB resistance including AC Sterling, Chevron, CI4196, Gobernadora, Harbin, Morrison, Siejo II, Symko, and Zhedar 1. Note that AC Sterling, Morrison and Symko are actually eastern Canadian varieties which we had not been using until recently. These 25 lines have shown consistently lower DON levels than AC Metcalfe over several years of testing in FHB nurseries. Initial results suggest that these gains may have been accompanied by trade-offs with resistance to other diseases like stem rust and spot blotch which are also important diseases in the FHB affected areas. These lines will be grown in advanced yield tests at multiple-sites in 2003 with the most promising being entered in registration tests in 2004.

Of particular note in the six-row and hulless barley breeding program at Brandon is the two-row hulless line EX645-3-6 which has low DON content and will be evaluated in the 2003 NABSEN test. A number of six-row lines, both hulless and covered, with low DON content have also been identified and will be advanced through the breeding program.

The Crop Development Centre / University of Saskatchewan barley breeding programs have identified and advanced many breeding lines with promising FHB resistance relative to AC Metcalfe and CDC Sisler. Some of these may eventually become new varieties in Manitoba and eastern Saskatchewan. Of particular interest are lines from the non-malting program from crosses involving CDC Freedom, CI4196 and HDE84194 as resistance sources. Some of these are being evaluated in yield tests.

It is expected that new two-row hulless, feed and malting barley varieties with improved FHB resistance will be registered over the next 5 years. The order of the classes listed above is probably the order in which the new varieties will be released with two-row malting barley being the most difficult and slowest because of quality constraints super-imposed on this problem. Improvements in FHB resistance, as indicated by lower DON levels, will probably be incremental in nature – no large reduction in DON content is expected in the near future. It may be 10 years before new six-row varieties with significantly lower DON levels than CDC Sisler are registered. However, it is possible that our American colleagues, who have been working diligently on improving FHB resistance in six-row malting barley for many years now, may develop such a variety sooner which may also be adapted to Manitoba.

We were successful in developing protocols for IVS that allowed us to regenerate DH plants, but the results from the field have been somewhat disappointing to date. IVS was applied to current varieties, resistance sources and segregating populations. The results were not unexpected for varieties and resistance sources, but it would only take the identification of one line from a popular variety with significantly lower DON content to make the effort worthwhile. Of particular interest are several lines derived from the six-row malting variety Excel which may have reduced DON levels. Further testing will be needed to confirm these results. One of the main reasons for using IVS on varieties and resistance sources was to develop protocols that could be successfully used in screening populations segregating for FHB resistance. Unfortunately, in vitro selection was successful only in 2001 for the two-row sub-population of the Chevron/CDC Fleet cross and in 2002 for the Rivers//Rivers/SB93806 breeding cross. However, only a limited number of protocols have been used in the segregating populations so far. It is possible that fine tuning these protocols, such as adjusting the concentration and composition of mycotoxins and length of exposure to the mycotoxins in culture, may improve results. Research is currently underway for that purpose, and has been extended to isolated microspore culture since the breeding program now utilizes this technique for routine DH production. If IVS is successful, it offers many advantages to the breeding program. However, even if IVS is not successful, lines identified with improved FHB resistance (from either IVS or control groups) will be advanced in the breeding program.

A number of DH populations have been developed for potential genetic studies on FHB resistance, but additional funding will be needed to proceed further. This is beyond the mandate of the current project.

We added a new objective in collaboration with the Crop Development Centre to develop a calibration for NIR to determine DON content more rapidly, easily and cheaply than is possible with current methods. Results to date are encouraging, but the accuracy of the equation in predicting DON content will need to be improved using more samples containing a wide range in DON values from different varieties grown at various locations over a period of time. This calibration has been developed for ground samples. An equation based on whole grain also shows promise but many more samples must be added to improve accuracy. This would eliminate grinding and make initial screening prior to ELISA significantly more efficient. The number of samples that can be analyzed for DON content is a bottleneck in improving FHB resistance in barley.

Utilizing a set of varieties evaluated over all three years of the project, we observed that the correlation coefficients for DON content (ELISA) and FHB ratings over years were moderate in magnitude, suggesting that progress is possible but it will be a long-term effort. Data on visual symptoms from the FHB nursery at Hangzhou, China, were weakly correlated with traits measured at Brandon.

Conclusion:

In conclusion, the funding provided by ARDI has laid the groundwork for developing new barley varieties with improved FHB resistance. This is expected to increase the selection of barley from the eastern prairie region for malting and brewing purposes in both domestic and export markets, particularly the lucrative American market which is readily accessible. Since malting barley receives a premium over feed barley, this would put more money in the pockets of producers and give them more cropping options. Producers of feed barley would benefit from improved levels of FHB resistance because their grain would be accepted locally by the livestock feeding industry and not downgraded for DON or shipped out of province at high costs. Thus, the profitability of growing feed barley would be increased. Improving FHB resistance would also reduce the need to use costly fungicides which are not 100% effective in controlling the disease. The feed industry, particularly the rapidly expanding hog industry, would also benefit from an assured supply of locally grown, uncontaminated barley, which would lower its costs and increase profitability. Continuing expansion of the livestock feeding industry will have many spin-off effects on the local economy of the eastern prairie region.

As a result of the project, we have identified the most FHB resistant barley varieties and advanced breeding lines currently available. Some of these advanced breeding lines may be under commercial production within 5 years, particularly for two-row barley. This would reduce susceptibility to FHB and, in combination with good agronomic practices, realize the above benefits under low to moderate levels of FHB. However, it may take up to 10 years for six-row varieties with lower DON content than CDC Sisler to be available commercially. The development of highly resistant barley varieties using exotic sources of resistance will take longer due to unfavourable effects on other important traits. Consequently, the full benefit will not likely be realized within 5 years of project completion.

Acknowledgements:

This project was made possible due to funding from the Governments of Manitoba and Canada through the Canada-Manitoba Agri-Food Research and Development Initiative (ARDI). We also wish to acknowledge financial support from the Western Grains Research Foundation Barley Check-off and Endowment Funds, Saskatchewan’s Agriculture Development Fund, and Agriculture and Agri-Food Canada’s Matching Investment Initiative program.