Fungicide Resistance – an increasing problem
Eugene O’Sullivan, Brendan Dunne, Steven Kildea, and Ewen Mullins
Teagasc, Oak Park Crops Research Centre, Carlow
Summary
Investigations of randomly-selected wheat crops in 2006 showed that strobilurin resistance in Septoria tritici was higher than it had been in 2003. This is despite a reduction in selective pressure for resistance due to reduced use of strobilurins for disease control in wheat in recent years.
Septoria populations remained sensitive to the triazoles Opus and Proline; the levels of sensitivity to these two products have not changed since 2003. There were shifts in the sensitivity of septoria to the triazoles Folicur and Caramba between 2004 and 2005 but no further reductions in sensitivity between 2005 and 2006. There is cross-sensitivity between Folicur and Caramba and spraying with either fungicide rapidly selects an insensitive septoria population which in turn reduces the efficacy of both fungicides. Septoria populations with reduced sensitivity to Folicur and Caramba are not less sensitive to Opus and Proline.
A new race of the barley leaf scald pathogen Rhynchosporium secalis emerged which caused severe disease on the previously resistant barley cultivar Doyen. Rhynchosporium populations in 2006 had the same levels of sensitivity to triazole and strobilurin fungicides as they had in 2003.
Studies of eyespot in wheat crops showed that the R type (Tapesia acuformis) is still the dominant strain and all populations are still predominantly resistant to MBC fungicides. There is also reduced sensitivity to Sportak and Unix.
Because of the occurrence of resistance to strobilurins the triazoles are now the most important group of fungicides that are available to cereal growers. Disease control must be managed in a way that minimises the potential for the build-up of insensitivity to these fungicides in fungal pathogens. Triazole fungicides should be mixed with suitable non cross-resistant partner fungicides such as chlothalonil or boscalid.
Introduction
Fungicides have been used to control plant diseases for over one hundred and fifty years. There were very few instances of resistance to fungicides until the introduction, over thirty years ago, and subsequent widespread use of systemic fungicides. The older fungicides that predated the systemics had both protectant and broad spectrum activity. They were multi-site inhibitors i.e. they interfered with a number of vital functions controlled by a number of genes in the target fungal cell. In order for resistance to develop to these products a number of specific genetic changes would have to occur at the same time in the target pathogen cell and the probability of this occurring is low. The systemic fungicides are usually directed against specific groups of pathogens. Many are single-site inhibitors i.e. they interfere with a vital function controlled by a single gene in the target pathogen cell. This makes it easy for resistance to develop since a single mutation in this gene can negate the effects of the fungicide.
Single genetic changes usually produce highly resistant strains of pathogens. A resistant strain may initially be present at a very low frequency in a pathogen population. It survives fungicide treatments and builds up rapidly, due to absence of competition from sensitive strains, to become the dominant component of the population and disease control fails. Increasing the rate of fungicide applied will not affect control. Resistant strains of pathogens may be less fit and at a competitive disadvantage compared with sensitive strains. In such cases the level of resistance will decrease rapidly if the fungicide concerned is withdrawn though it is likely to build up rapidly again if unrestricted use of the fungicide resumes. If there is not a fitness penalty resistance levels remain high indefinitely even if the fungicide concerned is no longer used.
Frequently a pathogen develops reduced sensitivity to a fungicide rather than complete resistance. Less sensitive strains of the pathogen are selected gradually through small cumulative changes induced by the fungicide. The effects on disease control are not as dramatic as those from complete resistance resulting from single genetic changes. Initially there may be only a slight decrease in sensitivity with no noticeable reduction of disease control. The continued use of the fungicide concerned leads to the selection of strains that are progressively less sensitive with corresponding reductions in fungicide efficacy. In such cases increasing the rate of fungicide may improve disease control.
Fungicide resistance has been recognised as a factor affecting the control of cereal diseases in Ireland since the 1980s when it became apparent that eyespot was no longer being controlled by MBC fungicides. Subsequent investigations showed that populations of the eyespot fungi (Tapesia yallundae and T. acuformis) had become predominantly resistant to these fungicides (Cunningham 1990). The field performances of some of the older triazole fungicides against Septoria have decreased over the years and this may be due to decreased sensitivity in populations of this pathogen, but there was no initial baseline data for sensitivity to these fungicides.
Baseline data on the sensitivity of some of the major cereal pathogens to the main groups of fungicides used to control them has been compiled at Oak Park since 2002. The results from each season’s sensitivity testing are compared with these baselines and in this way any shifts in the sensitivity of pathogens to the fungicides concerned can be detected.
Septoria Disease in Wheat
Strobilurin resistance in septoria
Resistance to strobilurin fungicides first emerged in Septoria tritici populations in Ireland in 2002 and by early 2003 resistance was found in populations in nearly all crops throughout the main wheat-growing regions of the country, usually at a very high frequency. Since 2003 there has been a dramatic reduction in the use of strobilurins in spray programmes on wheat crops, thus reducing the selective pressure for resistance. However, studies of S. tritici populations in fifteen selected crops throughout the wheat-growing regions of the country in 2004, 2005 and 2006 detected high levels of strobilurin resistance (Table 1). Levels of resistance in spring 2006, following four seasons of reduced usage of strobilurins, were even higher than they were in 2003 and 2004. So it appears that strobilurin resistance in septoria is genetically stable, does not carry a fitness penalty and will continue to remain high, even when the selection pressure is reduced.
|
Year |
Range of resistance |
Average |
|---|---|---|
| 2003 | 0% - 84% | 48% |
| 2004 | 50% - 100% | 83% |
| 2005 | 76% - 100% | 96% |
| 2006 | 86% - 100% | 97% |
Triazole sensitivity in septoria
Populations of S. tritici in 2003 were tested for sesitivity to epoxiconazole (Opus), which was the most widely-used triazole product at that time. In 2004, 2005 and 2006 populations were also tested for sensitivity to the triazole fungicides prothioconazole (Proline), tebuconazole (Folicur) and metconazole (Caramba) as well as to Opus. All isolates were sensitive to all fungicides in 2003 and 2004 and the levels of sensitivity detected then have been used as baselines against which sensitivity studies in subsequent years have been measured. There has been no shift in the sensitivity of S. tritici populations to Opus since 2003. All isolates tested up to and including 2006 had levels of sensitivity that were broadly similar to those detected in 2003 (Fig1). Levels of sensitivity to Proline have been similar to those for Opus with no shift in sensitivity between 2004 and 2006. There is evidence that when some triazoles are used extensively pathogens become less sensitive to them over time. Septoria is no more sensitive to the recently-introduced Proline than it is to Opus, which has been in use for many years, and this suggests cross-sensitivity between both of these triazoles.
In 2004 septoria populations were largely more sensitive to Folicur and Caramba, particularly to Caramba, where no isolates grew above 0.12ppm, than they were to Opus and Proline. There was a greater range in the sensitivity of isolates to Folicur. While less than 20% of isolates grew above 0.12ppm tebuconazole there were a few that grew up to 3.3ppm (Fig 2) and no isolates grew at this concentration in the case Opus, Proline or Caramba.
Fig. 1: Sensitivity of Septoria tritici isolates to epoxiconazole (Opus) 2003 – 2006
There was a shift in the sensitivity of septoria populations to Folicur from 2004 to 2005 with some isolates growing at concentrations of 10ppm tebuconazole and the proportions of isolates growing at the other concentrations towards the insensitive end of the scale (1.1ppm and 3.3ppm) increasing substantially compared with 2004 (Fig 2). While the proportions of these insensitive isolates of septoria increased in 2006 there was no further shift towards greater insensitivity i.e. no isolates grew above 10ppm tebuconazole which was the highest level of insensitivity detected in 2005. There was also a shift in sensitivity towards Caramba between 2004 and 2005 with isolates that were less sensitive to Folicur being also less sensitive to Caramba.
Fig. 2: Sensitivity of Septoria tritici isolates to tebuconazole (Folicur) 2004- 2006
Triazole insensitivity selection
In 2005 and 2006 all commercial winter wheat crops were sampled first in February-March, before any fungicides were applied and again in mid-July to determine whether or not the sensitivity of septoria might be affected by the fungicides used in disease control programmes. There were no changes in the sensitivity of septoria populations in any crops to epoxiconazole (Opus) or prothioconazole (Proline) between March and July in either year. This is despite the fact that most crops would have received products containing either or both of these fungicides as components of the various spray programmes used. However Folicur, applied usually at T3, selected septoria populations with reduced sensitivity to that product, and also to Caramba, in both years.
Figure 3 shows an example of a winter wheat crop in 2006 where no Folicur was applied. In this crop there was little change in the sensitivity of septoria to tebuconazole between March and July. Figure 4 shows an example of a crop where Folicur was applied and there was a marked increase in the proportion of insensitive septoria isolates between March and July. In the latter crop also the level of insensitivity to Caramba increased between March and July (Fig 5). This is despite the fact that Caramba had not been used as a component of the spray programme.
Fig. 3: Sensitivity of Septoria tritici isolates to tebuconazole in a commercial wheat crop where Folicur had not been applied
Fig. 4: Sensitivity of Septoria tritici isolates to tebuconazole in a commercial wheat crop where Folicur had been applied
Fig. 5: Sensitivity of Septoria tritici isolates to metconazole (Caramba) in a commercial wheat crop where Folicur had been applied
In other investigations on selection for triazole insensitivity, in 2004 and 2005 experimental plots were sprayed three times (T1, T2 and T3) with Opus, Proline, Folicur or Caramba at full, half and a quarter of the recommended rates. The results followed the same pattern in both years. Full or reduced rates of Opus or Proline did not select for insensitivity to either of these fungicides or to Folicur or Caramba. However, full or reduced rates of either Folicur or Caramba selected for insensitivity to both of these fungicides but not to Opus or Proline.
It is likely that reduced rates of fungicides would be less effective in controlling insensitive strains of pathogens than full rates and therefore would be more effective in selecting for insensitivity. However, this is not what has happened in the case of Folicur and Caramba. Whether the same is true for all triazoles is not clear since there have been no shifts in the sensitivity of septoria populations to Opus or Proline during the course of the present investigations.
Clearly, strains have developed in septoria that have reduced sensitivity to Folicur and Caramba and there is cross-sensitivity between these two fungicides. Spraying with Folicur or Caramba rapidly selects an insensitive population of septoria which in turn reduces the efficacy of these products. This has been borne out in Oak Park field trials in 2005 and 2006 where Folicur gave poor disease control compared with previous years.
Fortunately, a reduction in sensitivity to Folicur and Caramba has not affected sensitivity to Opus and Proline. Septoria isolates with reduced sensitivity to the former products are not less sensitive to Opus and Proline. So it appears that, unlike the strobilurins, reduced sensitivity or resistance to some triazole products will not affect all of these products.
The fact that there has been no shift in the sensitivity of septoria to Opus and Proline and that repeated spraying of plots with full or reduced rates of these has not so far selected less sensitive strains is reassuring. However, it cannot be taken for granted that resistance or a shift towards insensitivity will not occur and there must not be a return to the sense of complacency in the use of fungicides that there was before 2003. Disease control must be managed in a way that minimises the potential for the build-up of resistance and some guidelines are discussed later on in this paper.
Rhynchosporium in Barley
Leaf scald caused by Rhynchosporium secalis is a major disease of winter and spring barley in Ireland. Rhynchosporium overwinters predominantly on stubble debris or volunteer barley from previous crops but it can be carried on seed and transmitted from seed to seedlings (Kay and Owen 1973). Seed transmission is thought to be of minor importance as a source of primary infection though it can be important in long-range dispersal of the pathogen and also in the dispersal of new races. The disease is spread mainly by rain-splash dispersal of spores during the crop growing season. There is no known air-borne stage of the pathogen so resistance to fungicides in R. secalis should spread much more slowly than it did in the case of septoria. There are a number of different races of R. secalis and these vary in their virulence towards different barley cultivars.
Disease resistant barley cultivars can provide a cost-effective means of control. There are two types of resistance, single gene resistance and multigene resistance. The former gives almost complete disease control and is not affected by disease pressure or environment. However it is race-specific and can be overcome by the emergence of new races of the pathogen. Some highly resistant barley cultivars grown in New Zealand in the 1980s had their resistance eroded by the emergence of new races of rhynchosporium (Cromey, 1987).
Multigene resistance, also referred to as quantitative resistance or field resistance, only gives partial disease control but it is usually more durable than major gene resistance and not likely to be affected by new races. Partial resistance is affected by disease pressure and fungicide treatments are required to control rhynchosporium particularly when disease pressure is high.
A new race of R. secalis
The spring barley cultivar Doyen with high resistance to rhynchosporium had been widely grown in Ireland with no reports of severe disease. However in 2006 there were reports of disease epidemics in crops of this cultivar. Leaf samples were obtained from some of these crops in June and rhynchosporium was isolated from them. Cultivars, Lux, Tavern, Wicket and Doyen were inoculated with four rhynchosporium isolates from Doyen, and four isolates from other cultivars including one from 2003. These latter four isolates caused severe disease on Lux, Tavern and Wicket but practically no disease on Doyen. The isolates from Doyen however, caused severe disease on Doyen as well as in the other cultivars. This confirms that Doyen is highly resistant to the rhynchosporium population that is dominant on other cultivars in Ireland. The severe disease on Doyen in 2006 was not due to seasonal disease pressure but to the emergence of a race of R. secalis to which the cultivar has no resistance. This race may have been present in Ireland at a very low frequency and built up progressively in Doyen because of lack of competition from other races, or it may have been introduced recently from some external source.
Fungicide sensitivity in rhynchosporium
Previous investigations at Oak Park (2001 to 2003) showed that populations of R. secalis were generally sensitive to triazole and strobilurin fungicides with 20 to 30% of isolates resistant to MBC. Rhynchosporium populations were sampled again in 2006 to determine if there had been any shifts in sensitivity since 2003 and there was some concern lest the new race detected on the cultivar Doyen might also be resistant to fungicides. Rhynchosporium isolates collected during summer 2006 were tested for sensitivity to the triazole fungicides prothioconazole (Proline), epoxiconazole (Opus) and flusilazole (Sanction), the strobilurin azoxystrobin (Amistar) and to MBC (benomyl).
Of the triazole products, all isolates were most sensitive to Proline and were more sensitive to Opus than to Sanction (Fig 6). Rhynchosporium from Doyen had the same degree of sensitivity to all three triazole fungicides as that from other sources. Proline had not been included in previous resistance testing but the levels of sensitivity to Opus and Sanction are similar to those detected in 2003. Resistant isolates of R. secalis have been found in Scotland that can grow at 30 ppm epoxiconazole but no such isolates have yet been found in Irish populations.
Rhynchosporium from all sources was also sensitive to azoxystrobin with only 28% of isolates able to grow at concentrations up to 0.37% and none able to grow at higher concentrations. There has been no shift in sensitivity since 2003. Some 12% of isolates tested were resistant to MBC which is lower than the 20 to 30% resistance detected from 2001 to 2003.
Fig. 6: Sensitivity of Rhynchosporium secalis isolates to prothioconazole (Proline), epoxiconazole (Opus) and flusilazole (Sanction) 2006
Eyespot in Wheat
There are two different forms of the eyespot pathogen, differing in morphological characters and host range. Wheat or W-type isolates were pathogenic to wheat but caused little disease of rye, whereas Rye or R-type isolates were pathogenic to wheat and rye. Both W- and R-types are now recognized as separate species, Tapesia yallundae and T. acuformis respectively.
Populations of the eyespot fungi were preeviously investigated in Ireland in 1990. At that time the R type (Tapesia acuformis) predominated in all crops having replaced the previously dominant W type (T. yallundae) and all populations of eyespot were predominantly resistant to MBC-generating fungicides. MBC-generating fungicides have not been used to control eyespot since the 1980s.
Investigations of eyespot at Oak Park from 2001 to 2003 showed that there had been little change in species composition and fungicide resistance since 1990 (Table 2). The R type (T. acuformis) was still dominant but it had dropped from 89% of isolates in 1990 to 78% in 2003, with a corresponding increase in W-type (T. yallundae). MBC resistance was still as widespread in eyespot populations as it was in 1990, with over 90% of isolates resistant each year. This was despite the fact that MBC fungicides have not been used for eyespot control since the late 1980s. Reduced sensitivity to prochloraz was also widespread in eyespot populations occurring in 54% of isolates in 2003 compared with 31% in 1990. There was also reduced sensitivity to cyprodinil (Unix) in populations of eyespot fungi and this occurred in 24% of isolates in 2003.
|
Year |
No. of crops |
R type (T. acuformis) (%) |
W type (T. yallundae) (%) |
MBC resistance (%) |
Prochloraz reduced sensitivity (%) |
Cyprodinil reduced sensitivity (%) |
|---|---|---|---|---|---|---|
| 1990 | 37 | 89 | 11 | 87 | 31 | - |
| 2001 | 36 | 77 | 23 | 90 | 45 | 18 |
| 2002 | 76 | 72 | 28 | 91 | 52 | 27 |
| 2003 | 55 | 78 | 22 | 96 | 54 | 24 |
Eyespot populations in winter wheat crops were again sampled in 2005. The results indicated a further decrease in the frequency of the R type and an increase in the percentage of isolates with reduced sensitivity to Unix. As the number of crops sampled was low (15) and only a few isolates were obtained from some crops the results may not be an accurate reflection of the real situation. Sampling was carried out again in 2006 and testing of these samples is still in progress.
Managing Fungicide Resistance
The triazoles are the most important group of fungicides that are available to cereal growers. They are used to control many diseases of cereals. It is obviously in the best interests of all involved in crop protection – growers, advisors, researchers and agrochemical personnel – that these fungicides (and indeed all fungicides) are used in such a way as to maintain their effectiveness.
FRAC (Fungicide Resistance Action Committee) is a group comprised of government and industry scientists whose purpose is to provide fungicide resistance management guidelines to prolong the effective life of fungicides and to limit crop losses should resistance occur. The general guidelines provided by FRAC are:
- Limit the exposure of the pathogen population to the fungicide by reducing the number of applications in a growing season.
- Avoid using fungicides as eradicants.
- Avoid using multiple low doses and use high doses.
- Mix or alternate fungicides with different modes of action.
While these guidelines are admirable some are clearly impractical for instance the recommendation not to use fungicides in an eradicant situation. In many crops diseases, especially a disease such as septoria, are present from an early growth stage long before a fungicide is applied. The alternative is prophylactic treatments, which can often be uneconomic, or rely on some prediction of risk such as the use of Decision Support Systems which have yet to be fully validated.
There are mixed views on the effect of dose on selection of resistant strains of a pathogen. There are instances where high doses have resulted in greater selection pressure. However evidence that Septoria tritici in particular has become less sensitive to the triazoles over the last ten years suggests that high doses need be used for effective control of this disease. High doses mean a rate of fungicide close to or at the manufacturer’s recommended rates for their product. The use of mixtures or alternating fungicides with different modes of action is a commonly used strategy to prevent or delay resistance development. Mixtures can often give better disease control than single products.
As septoria is now resistant to the strobilurins, triazoles have become the foundation of fungicide programmes to control this disease. The current situation in regard to the sensitivity of the septoria population in Irish wheat crops to the triazoles has been discussed earlier in this paper. To maintain the current efficacy of the triazole group of fungicides to septoria they should be mixed with a suitable non cross-resistant fungicide. There are two such suitable products available in Ireland - chlorothalonil and boscalid.
Chlorothalonil is a broad spectrum multisite fungicide which is very active against septoria. It is a protectant fungicide and is used widely in mixtures with triazoles especially at the pre-T1 and T1 spray timings. In addition to reducing the potential for the development of insensitivity in pathogens, adding chlorothalonil to triazoles at the T1 spray timing reduces disease levels and increases yield as shown in Table 3. The response from the addition of chlorothalonil at T2 applications are less consistent but in general have been positive. There is a strong recommendation that if, at any spray application, a high level of eradicant activity is required (e.g. when a spray has been delayed) then it may be more beneficial to increase the triazole dosage and not to use chlorothalonil.
|
T1 Treatment |
Rate l/ha |
Yield t/ha @ 15% |
% Septoria |
|
|---|---|---|---|---|
|
Co. Meath |
Co. Cork |
2nd Leaf |
||
| Opus | 1.0 | 9.5 | 7.6 | 52 |
| Opus + Bravo | 1.0 + 1.0 | 9.9 | 8.0 | 35 |
| Proline | 0.65 | 9.3 | 8.1 | 37 |
| Proline + Bravo | 0.65 + 1.0 | 10.0 | 8.8 | 32 |
| Venture | 1.2 | 9.7 | 8.1 | 35 |
| Venture + Bravo | 1.2 + 1.0 | 9.9 | 8.7 | 30 |
Boscalid in contrast is a single-site protectant fungicide with a different mode of action to that of the triazoles. In this respect, as with chlorothalonil, it compliments and improves the action of the triazoles and reduces the possibility of resistance developing to the triazoles.. It is not available as a single product but in a preformulated mixture – Venture.
Chlorothalonil and boscalid are active against all strains of Septoria and should be used in mixtures with triazoles whenever they are being applied.
It was previously thought that as all triazoles had the same mode of action there would be cross-sensitivity among the various products. As discussed earlier, there is cross-sensitivity in Septoria between tebuconazole and metconazole but not between these products and epoxiconazole and prothioconazole. Prochloraz (Sportak) which has a common biochemical mode of action and cross resistance potential with the triazoles, has less efficacy against Septoria than the leading products. However, there is evidence from recent research in France that prochloraz may be effective on the strains of Septoria that have insensitivity to the other triazoles. In fact when either prochloraz or boscalid was mixed with epoxiconazole there was a decrease in insensitive isolates. Prochloraz may then be a candidate along with chlorothalonil and boscalid to be used as a mixing partner with other triazoles such as epoxiconazole and prothioconazole in a resistance management strategy. The benefits of prochloraz in this regard will be examined in trials in 2007.
Conclusions
- Resistance to strobilurin fungicides still remains high in septoria populations.
- Septoria populations remain sensitive to the triazole fungicides epoxiconazole (Opus) and prothioconazole (Proline).
- Septoria populations are less sensitive to the triazole fungicides tebuconazole (Folicur) and metconazole (Caramba) than they were in 2004.
- There is cross-sensitivity between tebuconazole and metconazole and spraying with either fungicide rapidly selects a septoria population with reduced sensitivity to both.
- A new race of the barley leaf scald pathogen Rhynchosporium secalis has emerged in Ireland.
- Rhynchosporium populations have the same levels of sensitivity to triazole and strobilurin fungicides as they had in 2003.
- Populations of eyespot pathogens are still predominantly resistant to MBC fungicides and some have reduced sensitivity to prochloraz and cyprodinil.
- Triazole fungicides should be used in mixtures with non cross-resistant partner fungicides in order to reduce the risk of resistance developing in the target pathogens.
References
- Cromey, M. G. (1987): Pathogenic variation in Rhynchosporium secalis in barley in New Zealand. New Zealand Journal of Agricultural Research 30: 95 – 99.
- Cunningham, P. C. (1990): MBC fungicide resistance in populations of the eyespot pathogen Pseudocecosporella herpotrichoide from intensively cropped cereal fields. Research Report, Crops Research Centre, Oak Park 179-180.
- Kay J. G. and Owen, H. (1973): Transmission of Rhynchosporium secalis in barley grain. Transactions of the British Mycological Society 60: 405 – 411.
Acknowledgement
The author would like to acknowledge the technical assistance of J. Grace in this work.
