Understanding fungicide resistance
Introduction
When a fungicide that is applied to a crop fails to control disease, control failure may be due to many causes, including poor sprayer calibration, operator error, excessive wind, washoff by rain, or an ineffective batch of fungicide product. It may also be because the disease has become resistant to the fungicide. Fortunately, fungicide resistance does not often arise, but there are some diseases and some fungicide chemicals where resistance occurs fairly frequently. Fungicides at risk from resistance are mainly the synthetic ones, developed since the 1970's, that are very selective in the way they affect their target fungi. The fungicide groups important in horticulture which are at risk are listed later in the article and include the benzimidazoles, dicarboximides, phenylamides and the DMI's. Many older fungicides, such as captan, copper, mancozeb, metiram, sulphur, thiram which are non-selective and have activity against a broad spectrum of diseases are not considered to be at risk from resistance, although instances of captan and copper resistance have been reported.
The first modern selective fungicide to suffer resistance was benomyl (Benlate) and others in the benzimidazole group. Yet surprisingly, benzimidazoles can still be very useful in many situations. One of the messages that came out of a recent International Fungicide Resistance Symposium in England was optimism regarding the continued usefulness of at-risk fungicides like benomyl. It was pointed out that there were no instances where a fungicide group had gone completely out of use as a result of resistance, and for many at-risk groups, resistance can be quite manageable.
Fungicide resistance can be a confusing business, because there are many concepts and terms which have very specific meanings in the context of fungicide resistance, but no meaning at all outside - a bit like computer jargon! However, because resistance is important to both orchardists and people in horticultural support industries it is essential that the main concepts and terms are understood. This article explains the essentials of fungicide resistance and outlines some of the issues and difficulties that are faced by fungicide resistance researchers.
How does resistance arise?
The first thing to understand is that fungicide resistance is a change in the disease-causing fungus, or the pathogen. It is not a change in the fungicide, nor in the host plant. The change in the pathogen from being sensitive to a fungicide to being resistant involves a genetic change which is passed on to successive generations of the fungus. To understand how resistance arises we must think of the pathogen in a crop as a population consisting of a mixture of strains which differ in their sensitivity to the fungicide. Some strains in the population may be so resistant that they cannot be controlled by normal application rates of the fungicide. Use of the fungicide therefore kills the sensitive strains but not the resistant ones, and over a period of time the resistant ones come to dominate. This is when a loss of disease control may occur. Fungicide chemicals do not actually cause resistant strains to form because they are not mutagens. Resistant strains, if they exist, arise from a very low natural rate of genetic mutation, and are initially present in the population at a very low level.
The word insensitivity means the same as resistance, although it is often used with a slightly different connotation. Because there are large amounts of money and peoples' livelihoods tied up in the fungicide chemical industry, and because growers depend on fungicides for economic returns, the word resistant is often too alarming to bandy about. The word resistant implies that the pathogen is either resistant or is not resistant. This does not reflect the way mixtures of sensitive and resistant strains affect disease control in crops. So rather than resistance, the word insensitivity is almost always used in practical discussions to give the idea of degrees of resistance. Unfortunately this can lead to convoluted statements like, strain A is less insensitive than strain B; meaning that strain A is controlled by the fungicide better than strain B. The word tolerance is also sometimes used instead of resistance. It shouldn't be, because tolerance is used for describing maximum limits for pesticide residues in food.
Detecting and studying resistance
To determine whether a fungicide is under threat from resistance, it is necessary to demonstrate resistance in the laboratory as well as a loss of disease control in the field. In many cases, loss of field performance is not obvious, and there can be difficulty in demonstrating a link between laboratory resistance and loss of disease control. Much fungicide resistance work is based on agar plate studies in the laboratory because this is often the only practical method of study.
In laboratory tests for resistance, it is necessary to isolate the pathogen and examine whether or not it can grow in the presence of the fungicide. This is usually done by culturing single spores of the fungus on agar plates. The fungal colonies which grow from these are called single spore isolates and represent individual genetic units from the population. When groups of isolates are found to have similar levels of resistance they are called strains, although the words strain and isolate are often used synonymously.
If you grow either sensitive or resistant strains on agar in the presence of a fungicide, the amount of growth decreases as the concentration of fungicide increases. A difference in resistance is a matter of the degree to which growth is inhibited. As the concentration of fungicide is increased, a point is reached at which the rate of growth is reduced to 50% of its rate in the absence of fungicide (Fig. 1). This concentration is the EC50 (50% effective concentration) or ED50 (50% effective dose) for a particular isolate. The EC50 is a means of comparing the reponses of different isolates to fungicide concentration. Fungicide concentration is expressed in mg/litre, which is the same as parts per million (ppm). It is important to note that the concentration refers to active ingredient (a.i.) of the fungicide. Fungicide products are a mixture of the active fungicide and other materials.
In describing the response of isolates to a fungicide, an EC95 value is sometimes also given. This is the concentration which inhibits growth by 95% compared to growth without fungicide. It provides additional information about growth over a wider range of fungicide concentrations. Another figure sometimes quoted is the minimum inhibitory concentration (MIC), which is the lowest concentration of fungicide which inhibits visible growth of the fungus. The MIC would be nearer to the EC95 than to the EC50.
Resistance occurs when the EC50 of an isolate is several times greater than the concentration that would normally inhibit growth. To determine what is normal, you need to know the baseline sensitivity, or the EC50 for strains that are controlled by the fungicide. The best baseline sensitivity information comes from strains that were around before the particular fungicide was ever used. This is referred to as the wildtype sensitivity. Since it is not possible to determine a true wild-type sensitivity after a fungicide has come into use, baseline sensitivity is often estimated using isolates from properties where there is no known history of use of the particular fungicide.
The number of times the EC50 of a resistant strain is greater than the baseline sensitivity is known as the resistance factor. In Fig. 1 the EC50 for isolate A is 0.26 mg/l and for isolate B it is 3.4 mg/l. The resistance factor for isolate B compared to isolate A is therefore 3.4 divided by 0.26, or approximately 13.
Once it has been established that there are resistant strains present, then the resistance frequency, or the percentage of isolates which are resistant, gives an indication of the likely effect on disease control. If resistance frequency is high, e.g. greater than 50%, then disease control problems are likely.
The EC50 is the essence of fungicide resistance research, yet it is actually quite a subjective thing to determine. Different EC50 values for a given fungal isolate can be obtained from different methods, e.g. inhibition of growth in agar culture compared to inhibition of spore germination. Even the ranking of EC50's for a number of strains can differ according to the method used.
How long does resistance persist?
If you stop using the fungicide will resistance disappear? The answer depends on the fungicide and the disease, but in many instances resistant strains do not survive well in the absence of the fungicide. This is because the genetic change that makes a strain resistant may make its biochemistry less efficient than a sensitive strain's when the fungicide is not around. The inability of resistant strains to compete is called a loss of fitness. It means that in the absence of the fungicide resistant strains die out and the resistance frequency decreases, allowing disease control to occur again.
The loss of fitness in resistant strains is the reason why resistance can be managed in some diseases by limiting the number of fungicide applications. Losses of fitness accompany dicarboximide resistance in Botrytis cinerea (grey mould) and in Monilinia fructicola (brown rot of summerfruit). In the case of B. cinerea resistance to benzimidazoles, resistant strains are just as fit as sensitive strains and persist even without use of the fungicide.
Cross resistance, dual resistance and mode of action
The mode of action of a fungicide is the specific way in which it inhibits the biochemistry of the target fungus. The genetic change in the fungus that gives it resistance to a fungicide will also give it resistance to other fungicides with the same mode of action. This is called cross resistance, and all the chemicals to which a strain is resistant are said to belong to the same cross resistance group.
The DMI fungicides are interesting because, although they are a chemically diverse group, they all have the same mode of action and there tends to be cross resistance among all of them. In surveys of resistance it is important to examine a number of related fungicides to gain a picture of the cross resistance characteristics of any resistant strains which may be found. In some cases resistance to one fungicide may increase sensitivity to another fungicide and this is called negative cross resistance.
It is possible to find resistance to more than one fungicide group in a single pathogen strain, e.g. strains of Botrytis cinerea in New Zealand can be resistant to both the benzimidazole and dicarboximide fungicide groups. This is called dual resistance (multiple resistance if it is to more than two fungicide groups). There is presumed to be separate genetic control of the resistance to each fungicide group.
Genetics of resistance
No matter how much you hate genetics, and most normal people do, you can't escape the need for genes, alleles, loci and other such things when trying to understand how resistant strains arise and survive. This is not the place to go into much detail, but a few points about the genetics of resistance are worth making.
The change in a resistant strain which allows it to grow in the presence of a fungicide might arise from either small changes to a number of genes which add together, or a change in a single major gene. In the cases of benzimidazole resistance and metalaxyl resistance in potato late blight, single major genes are involved. On the other hand, dicarboximide resistance, dodine resistance in the apple black spot fungus, and resistance in various fungi to DMI's, several independent genes are involved and in each case their effects add together.
Some information on the type of genetic control that causes resistance can be obtained by examining EC50 values for large numbers of isolates. If a group of isolates is found with distinctly higher EC50's than the rest of the population, then the resistance has probably been caused by a single gene mutation. Continued use of the fungicide will very rapidly cause the resistant strain to become predominant and this is called disruptive selection. For resistance that arises from additive minor genes, there tends to be a gradual shift in EC50 towards greater resistance. This is called directional selection. Monitoring of EC50's is very useful to warn of directional selection and strategies can be implemented to slow its development.
Many fungi undergo a sexual cycle each season. In the sexual cycle the genes can be reshuffled among strains and this gives the chance for new genetic combinations and the possible expression of resistance genes. The timing of fungicide applications in relation to the sexual cycle can be important. For apple black spot there used to be a recommendation to apply benomyl to fallen apple leaves to kill the fungus as it overwintered. Black spot undergoes sexual recombination in fallen leaves on the ground in winter. The resistance of black spot to benomyl which arose was possibly accelerated by the use of the fungicide when recombination of genes was occurring.
Managing resistance
Fungicide resistance is manageable where there is a loss of fitness with resistance. This occurs with dicarboximides for Botrytis cinerea and Monilinia fructicola, and in most cases of resistance to DMI's. The best strategy for slowing the appearance of resistance is to minimize use of the at-risk fungicide. In addition it has been proposed that tank mixing at-risk fungicides with broad spectrum nonselective fungicides, and also alternating applications of these types of fungicides, can slow the development of resistance. The usefulness of these strategies is at present a major topic of international debate. Field experiments using mixtures or alternations have been carried out but the results have been confusing. The best evidence that mixtures are effective in slowing resistance development appears to be for metalaxyl/mancozeb mixtures to combat resistance in potato late blight.
Are mixtures just chemical companies trying to sell more fungicide? The answer is probably no. Despite the difficulties in showing experimentally that mixtures slow down resistance development, there is general agreement among government and chemical industry researchers around the world that the use of mixtures is better than the use of at-risk fungicides alone. The reasoning is that the longer the pathogen is exposed to the at-risk fungicide without being killed, the greater is the risk of a resistant strain being selected. The pathogen must be knocked down and kept down to minimize its reproduction when the at-risk fungicide is present. Therefore mixing with a non-selective product improves the fungicidal effectiveness of the application and reduces the chances of a resistant strain emerging.
Another proposed strategy, that of frequent applications of the at-risk chemical at reduced application rates would appear to have exactly the wrong effect. Another suggestion has been to alternate applications of reduced rates of the at-risk fungicide with a non-selective fungicide. Again, any use of the at-risk fungicide that leads to it being present at concentrations that do not kill the fungus is asking for trouble.
Conclusions
Fungicide resistance causes disease management problems only where field resistance occurs and alternative fungicides are not available. The most important horticultural diseases at present affected in New Zealand are Botrytis diseases of grapes, berryfruit, kiwifruit and glasshouse crops, and brown rot of summerfruit. Should resistance to the DMI fungicides arise, the economic consequences could be serious. Of particular concern is resistance in apple black spot and in several powdery mildew diseases. Monitoring for shifts towards resistance is important and growers should seek advice as to whether resistance monitoring should be done on their properties.
In New Zealand we tend to rely on overseas research for information on which fungicides are at risk and how resistance should be prevented or managed. Some resistance monitoring surveys are being carried out in New Zealand, but the amount of research here is generally insufficient to deal with the threat that resistance poses. Fungicide resistance research relies on inputs from fungicide chemistry, biochemistry, genetics, plant disease epidemiology and disease management. The restructuring of New Zealand plant protection science into separate horticultural, arable and pastoral industry sectors has made it difficult to co-ordinate fungicide resistance research. This is because the questions which need answering cut right across sector boundaries.
Guidelines for resistance management are at present being reviewed by the New Zealand Committee on Pesticide Resistance (NZCPR), which is made up of industry, University and Crown Research Institute members. Guidelines were last reviewed in 1988-89 (Proc. 41st and 42nd N.Z. Weed and Pest Control Conferences) and the committee has reconvened in 1994-95 to update strategies for resistance prevention and management.
Fungicides at risk from resistance
The DMI's have become one of the most important groups of fungicides and are being closely watched for signs that resistance might develop. Overseas there are records of problems in powdery mildews of cereals and grapes, and in the apple black spot fungus. DMI's have many desirable features, including high fungicidal activity, low toxicity to other organisms, protective and curative properties and compatibility with integrated pest management. They are a chemically diverse group which all inhibit the same demethylation step in the biosynthesis of ergosterol, a vital component of cell walls in many fungi. The term ergosterol biosynthesis inhibitors (EBI's) is also used for DMI's. However, EBI is a broader grouping and includes morpholine fungicides, which inhibit a different step in the ergosterol biosynthesis pathway.
The DMI's used in horticulture include triforine (Saprol) which belongs to the piperazine group, pyrifenox (Dorado) a pyridine, fenarimol (Rubigan) a pyrimidine, imazalil (Fungaflor) and prochloraz (Octave, Sportak) which are imidazole fungicides. By far the biggest group are the triazoles, and common ones include bitertanol (Baycor), cyproconazole (Alto), flusilazol (Nustar), myclobutanil (Systhane), penconazole (Topas), propiconazole (Tilt), triadimefon (Bayleton) and triadimenol (Cereous).
Because DMI's are used for many diseases in many crops and there are only a few cases of resistance, the risk of resistance can be considered to be generally low. One difficulty in monitoring resistance to DMI's is the wide natural variation in the sensitivity of many pathogens. Isolates with quite different EC50's may not represent genetically different strains, but may be parts of a continuous population with variable sensitivity conditioned by the environment. For this reason surveys for DMI resistance must examine large numbers of isolates to gain an accurate indication of shifts in sensitivity.
Benzimidazoles
Benzimidazoles commonly used in horticulture include benomyl (Benlate), carbendazim (Bavistin, Delsene), thiabendazole (Tecto), thiophanate-methyl (Topsin, Taratek). This was the first group of selective fungicides for which resistance in plant pathogens was found, and benzimidazoles are now not effective for many important diseases.
Resistance to benzimidazoles is very persistent, and once it has arisen it tends to be there for good. However, the benzimidazoles are still important and useful, particularly for one-off applications where there is not the need for routine use. All have same mode of action, which is quite well understood, and resistance is controlled by a single major gene.
Dicarboximides
The dicarboximides, which include chlozolinate (Serinal), iprodione (Rovral), procymidone (Sumisclex) and vinclozolin (Ronilan), have activity against a limited number of fungi contained in nine genera. The most important is Botrytis cinerea, the cause of grey mould in many fruit, vegetable and glassshouse crops. Other uses include brown rot of stone fruit, white rot of onions and garlic, sclerotinia diseases and some turf diseases. These fungicides have low phytotoxicity, high fungicidal activity and short withholding periods. Although there are claims of some penetration of leaves and fruit and hence some curative activity, dicarboximides essentially have only protective activity.
Dicarboximide resistance has been most studied in Botrytis cinerea, and in New Zealand, resistant strains are widespread in grapes, kiwifruit and glasshouse crops. Resistance is accompanied by a loss of fitness and when use of dicarboximides stops, resistance frequency decreases. A limit of 1-2 applications per season makes resistance manageable. Loss of disease control has been more obvious in glasshouse situations than out-doors, but there is evidence that high resistance frequencies are associated with a loss of disease control. There are recent data that suggests resistant strains which are more fit are emerging. There is cross resistance among all the dicarboximides.
Dodine
Resistance of the apple black spot fungus to dodine has been reported overseas, but not in New Zealand. Dodine resistance is unusual because this fungicide is not selective in its mode of action, so would not be thought of as being at risk from resistance. Where resistance has appeared it has been a gradual decrease in sensitivity controlled by several genes whose effects add together. It is important to monitor black spot in New Zealand for signs of resistance. There appears to be no information on whether there is a loss of fitness associated with dodine resistance.
Phenylamides
These fungicides include several chemical groups, the most important being the acylalanines, which include benalaxyl (Galben), furalaxyl (Fongarid) and metalaxyl (Apron, Ridomil, Speargard). They are active against the group of fungi called oomycetes, which include downy mildews, potato late blight, damping-off pythium fungi and phytophthora root rots.
Metalaxyl is the most active of the phenylamides. It has curative as well as protective activity, and rapid uptake and translocation to new growth. Resistance has occurred in a number of fungi, the best known being the potato late blight fungus. Resistance arose because metalaxyl was used intensively before the dangers of resistance were understood. Mixing metalaxyl with mancozeb slows down the build-up of resistance. Resistance was thought to be persistent, but recent evidence from Ireland suggests that it is associated with a loss of fitness and can be managed by limiting the number of applications. The phenylamide fungicides form one cross resistance group.
Morpholines
Morpholines include fenpropimorph (Merit) and tridemorph (Calixin). They are mostly used in cereals for powdery mildew and rust control, but also for powdery mildew in cucurbits and ornamentals, and in bananas for leaf streak diseases. Like the DMI's, they are ergosterol biosynthesis inhibitors (EBI's), but they inhibit a different biochemical step. Also like the DMI's, there is wide variation in base-line sensitivity and much debate as to whether or not resistance is developing. If resistance is developing, then it is a directional shift and monitoring of EC50's will give a valuable warning of a potential problem. Most research into morpholine resistance is being done in cereal powdery mildews in Europe.
Acknowledgement
This article was made possible through the New Zealand Fruitgrowers Federation Charitable Trust which funded my attendance at the Fungicide Resistance Symposium at Reading University in England in March 1994.