Last month marked the official launch of Revystar XE – a fungicide containing a shiny new azole, Revysol, and BASF’s flagship SDHI, Xemium. CPM takes a look at the evolution of fungicide insensitivity that the new chemistry will play a part in managing.
A remarkable group of chemistry.
By Lucy de la Pasture
The last new azole to hit the UK market was prothioconazole in 2005 but the fungicide development team at BASF have just written a new chapter to a book that most people thought was finished. Prof John Lucas of Rothamsted Research describes the discovery as ‘courageous and an important addition to the fungicide portfolio for a number of reasons.’
“The first cereal azole, triadimefon (Bayleton) was introduced into the UK in 1976, more than forty years ago, and there were subsequent generations of azoles which arrived throughout the seventies, eighties and early nineties,” he explains.
Tebuconazole, epoxiconazole and metconazole were in this last wave of chemistry and brought a marked improvement in disease control over the azoles that had gone before them. The arrival of a new group of chemistry, the Qols (strobilurins), seemed to outclass the performance of the azoles and the development race effectively stopped.
Fast forward a decade and septoria (Zymoseptoria tritici) populations in the UK and Ireland were starting to show a notable increase in the frequency of isolates with reduced sensitivity to azoles and the strobilurins had failed altogether. This was the backdrop to the launch of a new azole prothioconazole from Bayer.
“Prothioconazole was a big step forward, not just because of its efficacy on septoria, but for the other diseases it had activity on, such as eyespot,” highlights John. “It also proved that you can find new and desirable properties in a well-explored area of chemistry, and that azoles still had much to offer,” he comments.
“The arrival of a new generation of SDHI fungicides for use in cereals a few years later meant few people expected further new fungicides to emerge from the azole class of chemistry.”
John describes the azoles as ‘a remarkable group of chemistry’ which has provided a multitude of fungicides for use in agriculture, where the target site is the fungal sterol-14α-demethylase enzyme, commonly known as CYP51. But azole molecules have been equally as effective as human medicines and, ironically, it’s their success in both of these fields that has led to the gradual removal of many azole fungicides from the market.
In humans, azoles can be used for the treatment of oestrogen-dependent diseases, such as in breast cancer therapy. Whereas the inhibition of CYP19 (aromatase) is the working principle for tumour therapy, it’s an unwanted side effect of azoles used as fungicides or antifungal drugs and it’s this possibility ‘dual-activity’ that led to the concerns over endocrine disruption (ED) when the EU moved to a hazard-based system for the registration of agrochemicals.
With epoxiconazole under risk from the ED criteria, BASF set out to screen azole molecules to find one that didn’t act on CYP19 so wouldn’t have any endocrine disrupting risk. And as a result, the UK now has the first new azole to gain registration as a fungicide for 15 years and it has come at a very interesting time in terms of fungicide resistance management.
“The textbook says that once a fungicide class has been compromised by the development of resistance, all compounds in that class will most likely be affected. We now know that this is an over-simplification, and the azoles are a prime example,” explains John.
“By comparison with other single-site inhibitors, like the MBCs and QoIs, resistance to azoles has developed relatively slowly, and even when present, it doesn’t seem to impact all compounds equally.
“CYP51 has turned out to be not only an excellent target to inhibit, but also a highly conserved protein with tight constraints on what mutations are tolerated. Over the years we’ve seen gradual erosion of efficacy in pathogens like Z. tritici as CYP51 has changed over time in response to selection by fungicides and other resistance mechanisms accumulate, such as efflux – where the azole is pumped out of the cell before it can affect the target site – and over-expression, where the increase in the amount of CYP51 means more fungicide is needed to inhibit it. But no complete breakdown in efficacy has occurred.”
ADAS principal research scientist, Jonathan Blake presented AHDB’s fungicide performance data at its Agronomist Conference which took place last month. He believes that the results indicate that we’ve now reached the point where there’s been little further evolution in the septoria population, according to the results of Rothamsted Research early season monitoring in 2019, with a possible plateau in the ‘older’ azole activity now reached.
In the wider context, BASF monitoring across Europe has detected a clear and continued shift in the sensitivity of the septoria population to prothioconazole. Dr Rosie Bryson, BASF’s senior principal scientist, explains that dry seasons, such as 2017, seem to trigger a bigger shift in sensitivity the following year, possibly because more of the ‘fitter’ pathogens survive the adverse conditions.
“Up until now, over 60% of the fungicides applied to the wheat crop across Europe have contained either prothioconazole or epoxiconazole. We’ve identified cross-resistance between the two azoles, mostly this is found in Ireland and the UK because the weather conditions favour septoria development, putting the septoria population under higher selection pressure because of fungicides needed to control epidemics.”
Rosie explains that when talking about insensitivity to azoles it’s mutations at their target site, CYP51, in the septoria pathogen that are most often referred to. In the field the reality is that the situation is very much more complicated.
“Each septoria isolate with insensitivity to an azole has a complex of CYP51 mutations, with nine main haplotypes representing 85% of the European population.
“Each mutation effectively changes the shape of the target site which makes it more difficult for the fungicide molecule to bind, meaning it can’t as effectively inhibit the CYP51 enzyme. Even with complex haplotypes, such as G1 which has seven mutations, some of them will have a smaller effect than others, but generally the more complex they are then the less sensitive the septoria isolate is to fungicides,” explains Rosie.
This high level of background insensitivity in the septoria population makes it a challenging time to launch a new azole fungicide. “When epoxiconazole was released the septoria population was still sensitive and 15 years ago when prothioconazole came along, the population had probably begun to shift but was very different than the make-up of the population today,” she says.
It begs the question, what makes Revysol (mefentriflucanazole) different? Rosie explains that the new molecule belongs to a new class of azoles and is an isopropanol. The isopropanol “linker” on which the azole is attached gives Revysol’s chemical structure a shape that’s different to all the other azole molecules and a unique ability to change spatially.
“In the Revysol molecule, the triazole ring sits on the ‘neck’ of a flexible isopropanol unit. This unique chemical constellation allows the molecule to assume different conformations easily – bound and unbound.”
When Revysol ‘docks’ on to CYP51, it switches from the unbound to the bound form (which resembles a ‘hook’) and binds to the target enzyme up to 100 times more powerfully than conventional azole fungicides. It’s this ability to adapt and be flexible that makes Revysol capable of coping with the small changes in shape at the CYP51 target site caused by mutations.
“Its flexibility means it’s able to adapt its shape to bind with CYP51 even where the most complex target site mutations have developed,” explains Rosie.
The table shows the nine most common Zymoseptoria tritici haplotypes found in the European population and the CYP51 mutations associated with them. Source: Huf et al., 2019.
As a result, Revysol has consistently been performing at a level above the current best-performing azole, prothioconazole, in field trials and is obtaining a similar level of septoria control to that expected from the ‘newer’ azoles when they first came on to the market, before insensitivity evolved.
“In glasshouse work we’ve found Revysol can control all of the septoria haplotypes currently found across Europe,” adds Rosie.
Asked whether this strong level of performance can be expected to continue, Rosie says she’d love to be able to give a definitive answer to the question. The truth is no-one knows what will happen and John believes Revysol is highly unlikely to escape the evolution of some sort of resistance within some pathogen populations.
However, both scientists agree that the industry is far better informed in recent years and have learned lessons from the active ingredients that have succumbed to fungicide resistance. One of those is to protect it with another mode of action with similar efficacy and this is why Revystar XE contains the SDHI, Xemium (fluxapyroxad), deemed essential for sustainable resistance management.
Even though SDHIs have seen some erosion in efficacy over the past three seasons, they’re still giving good levels of performance in the field. Rosie believes it’s likely their activity will slowly erode further in time, but in 2019 there appeared to be a much smaller shift than happened from 2017 to 2018.
“There’s a wide perception in the field that SDHIs didn’t work as well in 2019, compared with 2018, but what’s actually happened is that the effect of the shift in sensitivity that happened in 2017 wasn’t really noticed in 2018 because it was so dry, and consequently the disease pressure was low.
“BASF insensitivity testing across Europe shows there’s been no statistical change in sensitivity from 2017 to 2019 across Europe but it identifies a higher level of variability, with the greatest range of mutations present in the UK and Ireland and declining in an eastward direction across the continent,” explains Rosie.
Since the first changes in septoria sensitivity to the SDHIs, detected in 2016, the evolution has been characterised by a series of target site mutations giving partial levels of resistance and a slow decline in efficacy rather than the step-change in activity that happened with the Qols in 2003.
“In monitoring, we’ve found the two most dominant mutations are C-T79N and C-N86S, both are moderately adapted. The severe C-H152R mutation has only ever been found to be present at very low levels in the spring so it does seem to have a fitness penalty which means it doesn’t overwinter well,” she explains.
BASF pathotype monitoring in UK, spring 2019
UK early season monitoring, n=44 (up to end of April)
Source: BASF, 2019.
The azole and SDHI components of Revystar have been balanced to produce a fungicide which can perform at a level greater than could be expected from the sum of its parts, adds Jonathan. He’s conducted Matrix trials at ADAS which explore the dose rates of each active ingredient alone and in mixture to identify whether there are any antagonisms or synergies between them.
“Using some complex mathematics, it’s possible to compare the observed performance of Revystar against its predicted performance. It’s a very tough test and it’s very unusual to find a fungicide that gives a better than predicted performance,” explains Jonathan.
“In 2018 we found Revystar performance almost perfectly fitted the line of prediction and in 2019, it exceeded it. That indicates there is a possible synergy between Revysol and Xemium, and Revystar may perform as well, or better than you’d expect from the sum of its parts,” he says.
Although the arrival of Revystar is undoubtedly an exciting development and will provide a much-needed option in a fungicide market where active ingredients are being lost at an alarming rate, it’s important not to underestimate the importance of stewardship in preserving its activity, believes John.
“We’re not going to get out of an evolutionary arms race with the septoria pathogen, but we are getting smarter about co-formulation, as Revystar demonstrates. We also have to be smart about how we use chemistry and remember Revystar will only take us back to where we were in terms of fungicide efficacy,” he says.
Integrated pest management will also play a crucial part in Revystar stewardship, says Rosie, with the adoption of more septoria-resistant varieties central to an overall resistance management strategy.
“It’s going to be just as important to try and hit the right timings in the field, even though Revystar has good curative activity, and to dose appropriately by matching the dose to variety susceptibility and disease pressure,” she advises.
John agrees selecting varieties with more durable resistance and both smarter formulation and use of chemistry are important tools to slow the development of fungicide resistance. He’s witnessed many classes of fungicide come and go over the years and warns, “the behaviour of the microbial world won’t change, even after Brexit!”
