FUNGICIDE RESISTANCE
`IN CROP PATHOGENS:
`HOW CAN IT BE MANAGED?
`
`2nd, revised edition
`
`KEITH J BRENT and DEREK W HOLLOMON
`
`

`

`Cover:
`Scanning electron
`micrograph of 7-day-old
`colony of powdery
`mildew
`(Blumeria graminis f.sp.
`tritici) on a wheat leaf.
`Insert shows a
`2-day-old colony at
`higher magnification.
`Although the sensitivity
`of mildew populations
`towards certain
`fungicides has changed
`considerably over
`the years,
`implementation
`of resistance
`management strategies
`has helped to sustain
`an overall satisfactory
`degree of control.
`(Syngenta)
`
`FUNGICIDE RESISTANCE ACTION COMMITTEE
`
`a Technical Sub-Group of
`
`CROPLIFE INTERNATIONAL
`
`Avenue Louise 143, 1050 Brussels, Belgium
`Telephone: + 32 2 542 04 10. Fax: +32 2 542 04 19
`
`www.frac.info
`
`

`

`FUNGICIDE RESISTANCE IN CROP PATHOGENS:
`HOW CAN IT BE MANAGED?
`
`FUNGICIDE RESISTANCE
`IN CROP PATHOGENS:
`
`HOW CAN IT BE MANAGED?
`
`KEITH J BRENT
`
`St Raphael, Norton Lane,
`Chew Magna, Bristol BS18 8RX, UK
`
`DEREK W HOLLOMON
`
`School of Medical Sciences
`Department of Biochemistry
`University of Bristol, University Walk, Bristol, BS8 1TD, UK
`
`Published by the Fungicide Resistance Action Committee 2007
`
`FRAC Monograph No. 1 (second, revised edition)
`
`ISBN 90-72398-07-6
`Dépot Légal: D/1995/2537/1
`
`Design and production by Newline Graphics
`Reprinted 2007
`
`1
`
`

`

`FUNGICIDE RESISTANCE IN CROP PATHOGENS:
`HOW CAN IT BE MANAGED?
`
`CONTENTS
`
`Summary
`
`Introduction
`
`Chemical Control of Crop Disease
`
`Defining Fungicide Resistance
`
`Occurrence of Resistance
`
`Origins of Resistance
`
`Resistance Mechanisms
`
`Monitoring: Obtaining the Facts
`
`Assessing the Risk
`
`Management Strategies
`
`Implementation of Management Strategies
`
`Benzimidazoles
`
`Phenylamides
`
`Dicarboximides
`
`SBIs (Sterol Biosynthesis Inhibitors)
`
`Anilinopyrimidines
`
`Qols (Quinone Outside Inhibitors)
`
`CAAs (Carboxylic Acid Amides)
`
`Resistance Management in Banana Production
`
`The Future
`
`Acknowledgement
`
`References
`
`Page No.
`
`3
`
`5
`
`6
`
`7
`
`9
`
`13
`
`16
`
`18
`
`23
`
`27
`
`34
`
`36
`
`37
`
`39
`
`40
`
`42
`
`42
`
`44
`
`45
`
`46
`
`50
`
`50
`
`2
`
`

`

`FUNGICIDE RESISTANCE IN CROP PATHOGENS:
`HOW CAN IT BE MANAGED?
`
`SUMMARY
`
`This publication gives a broad overview of efforts world-wide to combat
`problems in crop protection that are caused by development of resistance to
`fungicides. The following major points are emphasised:
`
`• Fungicide treatments are, and will remain, essential for maintaining healthy
`crops and reliable, high-quality yields. They form a key component of
`integrated crop management, and their effectiveness must be sustained as long
`as possible.
`
`• Pathogen resistance to fungicides is widespread. The performance of many
`modern fungicides has been affected to some degree.
`
`• Resistance problems could be much worse. All types of fungicide are still
`effective in many situations. Current countermeasures are by no means
`perfect, but they have proved to be necessary and beneficial.
`
`• Resistance builds up through the survival and spread of initially rare mutants,
`during exposure to fungicide treatment. This development can be discrete
`(resulting from a single gene mutation) or gradual (considered to be
`polygenic). Resistance mechanisms vary, but mainly involve modification of
`the primary site of action of the fungicide within the fungal pathogen.
`
`• Resistance risk for a new fungicide can be judged to some degree. High risk
`indicators include: single site of action in the target fungus; cross-resistance
`with existing fungicides; facile generation of fit, resistant mutants in the
`laboratory; use of repetitive or sustained treatments in practice; extensive
`areas of use; large populations and rapid multiplication of target pathogen; no
`complementary use of other types of fungicide or non-chemical control
`measures.
`
`• Monitoring is vital, to determine whether resistance is the cause in cases of
`lack of disease control, and to check whether resistance management
`strategies are working. It must start early, to gain valuable base-line data
`before commercial use begins. Results must be interpreted carefully, to avoid
`misleading conclusions.
`
`3
`
`

`

`FUNGICIDE RESISTANCE IN CROP PATHOGENS:
`HOW CAN IT BE MANAGED?
`
`• The main resistance management strategies currently recommended are: avoid
`repetitive and sole use; mix or alternate with an appropriate partner fungicide;
`limit number and timing of treatments; avoid eradicant use; maintain
`recommended dose rate; integrate with non-chemical methods. Wherever
`feasible, several strategies should be used together. Some are still based
`largely on theory, and further experimental data are needed on the underlying
`genetic and epidemiological behaviour of resistant forms, and on effects of
`different strategies. Lowering dose may not be adverse in all circumstances.
`
`• The industrial body FRAC has been remarkably effective in its essential and
`difficult role of coordinating strategy design and implementation between
`different companies that market fungicides with a shared risk of cross-
`resistance. Education and dissemination of information on resistance have also
`been valuable activities. New types of fungicide continue to appear, and
`receive close attention by FRAC.
`
`• Much research and formulation of advice on fungicide resistance have been
`done by agrochemical companies. Public-sector scientists and advisers also
`have contributed greatly to resistance management, in research and practice.
`Their liaison with industry has been generally good, and there are
`opportunities for further interaction.
`
`• The sustained supply of new and diverse types of chemical and biological
`disease-control agents, and their careful introduction, are seen as key anti-
`resistance strategies. This aspect of product development is now increasingly
`recognised by national and international registration authorities, many of
`which now require from applicants detailed information on the actual or
`possible occurrence of resistance, on base-line data, and on proposed
`monitoring activities and instructions for use.
`
`4
`
`

`

`FUNGICIDE RESISTANCE IN CROP PATHOGENS:
`HOW CAN IT BE MANAGED?
`
`INTRODUCTION
`
`‘A mutable and treacherous tribe’ – this apt description of the fungi was written
`by Albrecht von Haller in a letter to Carolus Linnaeus, ca. 1745.
`
`For some 35 years now the agricultural industry has faced problems arising from the
`development of resistance in fungal pathogens of crops, against the fungicides used to
`control them. Since the first cases of widespread resistance arose, agrochemical
`manufacturers, academic and government scientists, and crop advisers, have put a
`great deal of effort into analysing the phenomenon and establishing countermeasures.
`In 1994 the Fungicide Resistance Action Committee (FRAC), now affiliated to
`CropLife International, commissioned a broad review of progress world-wide in
`dealing with fungicide resistance, and of the outstanding difficulties that need to be
`overcome.
`
`This was published as FRAC Monograph No 1 (Brent 1995). The key tenets of
`resistance management have not changed over the intervening years, but there have
`been many developments in fungicide chemistry, in the incidence of fungicide
`resistance, in knowledge of resistance mechanisms, and in resistance management
`projects. As far as possible these have been incorporated into this Second Edition. As
`before, this publication aims to be an informative article for all who are concerned
`professionally with crop disease management, including biologists, chemists,
`agronomists, marketing managers, registration officials, university and college
`teachers, and students. It is meant to be read, or at least skimmed, as a whole. It is not
`intended as a detailed work of reference for the specialist, although a limited number
`of literature citations, out of the several thousand publications on this topic, are
`provided for those readers with a deeper interest. Earlier reviews concerning fungicide
`resistance management (Dekker, 1982; Brent, 1987; Schwinn and Morton, 1990;
`Staub, 1991) were drawn upon freely in the original preparation of this monograph
`and are still of considerable value. A review paper by Kuck (2005) has provided more
`recent information and comment. Where appropriate the authors have endeavoured to
`discuss differing viewpoints, but conclusions are theirs and do not necessarily reflect
`the views of FRAC.
`
`5
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`

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`FUNGICIDE RESISTANCE IN CROP PATHOGENS:
`HOW CAN IT BE MANAGED?
`
`Two further FRAC Monographs (No 2, Brent and Hollomon 1998; No.3, Russell,
`2003), respectively address in more detail two major components of fungicide
`resistance management: the assessment of risk, and the establishment of sensitivity
`baselines. A second, revised edition of Monograph No. 2 is available.
`
`CHEMICAL CONTROL OF CROP DISEASE
`
`Fungicides have been used for over 200 years to protect plants against disease attack
`by fungi. From small and primitive beginnings, mainly to protect cereal seeds and
`grape-vines, the number of crops and crop diseases treated, the range of chemicals
`available, the area and frequency of their use, and the effectiveness of treatments, have
`increased enormously, especially since the second world war.
`
`Remarkably, two very old-established remedies, copper-based formulations and
`sulphur, are still used widely and effectively. Several ‘middle-aged’ fungicides
`(phthalimides, dithiocarbamates, dinitrophenols, chlorophenyls) have been used
`steadily for well over 40 years. A large number of more potent fungicides, of novel
`structure and mostly with systemic activity not found in the earlier products, were
`introduced in the late 1960s and 1970s. These included 2-amino-pyrimidines,
`benzimidazoles, carboxanilides, phosphorothiolates, morpholines, dicarboximides,
`phenylamides, and sterol demethylation inhibitors (DMIs). Introductions in the 1980s
`mainly were analogues of existing fungicides, particularly DMIs, with generally
`similar though sometimes improved properties. Over the past decade, however, a
`number of novel compounds have been introduced commercially or have reached an
`advanced stage of development – these include phenylpyrroles, anilinopyrimidines,
`quinone outside inhibitors (QoIs, including strobilurin analogues), benzamides and
`carboxylic acid amides
`
`The more recent fungicides are generally used in relatively small amounts, because of
`their more potent action against plant pathogens. However, their margins of safety to
`mammals and other non-target organisms are no smaller and are often greater, when
`compared weight-for-weight with those of the older materials.
`
`Spraying has always been the principal method of fungicide application, and the
`conventional hydraulic sprayer still predominates. Reduction in spray volume, and
`more stable and safer formulation, are probably the most significant advances that
`
`Modern spraying of
`fungicides in cereal
`fields in Europe.
`Use of wide spray
`booms and ‘tram-lines’
`aid timely and precise
`application, but the
`continued effectiveness
`of the fungicides
`themselves is a more
`basic requirement.
`(FRAC).
`
`6
`
`

`

`FUNGICIDE RESISTANCE IN CROP PATHOGENS:
`HOW CAN IT BE MANAGED?
`
`have been made in application technology. The frequency and timing of spraying have
`not changed a great deal from early recommendations, although the advent of the
`systemic fungicides has permitted some greater latitude in these parameters and has
`increased the feasibility of using disease threshold or forecast approaches. Roughly
`half of the crop diseases treated require treatment only once or twice per season, and
`the remainder require three or more (in some cases up to 20) applications. Systems of
`integrated crop management involving minimum necessary chemical and energy
`inputs, and use of complementary non-chemical protection measures wherever
`possible, have been widely adopted and to some extent have led to a reduction in spray
`number and dose in some situations.
`
`At present some 150 different fungicidal compounds, formulated and sold in a several-
`fold larger number of different proprietary products, are used in world agriculture. The
`total value of fungicide sales to end-users is approximately 7.4 billion US dollars
`(source: Phillips McDougall, Industry Overview, 2005). Nearly half of the usage is in
`Europe, where fungal diseases cause the most economic damage to crops. Most of the
`recommended treatments generally provide 90% or greater control of the target
`disease, and give the farmer a benefit: cost ratio of at least 3:1. Some diseases, e.g.
`wheat bunt caused by Tilletia spp. or apple scab caused by Venturia inaequalis, require
`an extremely high level of control for various commercial or biological reasons. For
`some others, e.g. cereal powdery mildews (Blumeria graminis), the risks associated
`with somewhat lower standards of control are smaller. Some fungicides control a
`rather wide range of fungal diseases, whereas others have a limited spectrum of
`activity against one or two specific groups of plant pathogens. Although many
`fungicides are marketed, any one major crop disease typically is well controlled by
`only three or four different types of fungicide, so that any fall in effectiveness of a
`previously reliable fungicide through resistance development can be a very serious
`matter for the grower.
`
`DEFINING FUNGICIDE RESISTANCE
`
`A potential new fungicide is identified in laboratory and glasshouse tests on different
`types of fungal pathogen, and is then tested in field trials against an appropriate range
`
`7
`
`

`

`FUNGICIDE RESISTANCE IN CROP PATHOGENS:
`HOW CAN IT BE MANAGED?
`
`of crop diseases in different regions and countries. Only if it works uniformly well
`against important crop diseases in a large number of trials over several seasons is it
`considered for development and marketing. The pathogens it works against are
`deemed to be ‘sensitive’, and those that it does not affect or hardly affects are regarded
`as ‘naturally’ or ‘inherently resistant’. This pre-existing type of resistance is of no
`further practical interest once it has been identified as a limitation to the range of use
`of the fungicide. Reasons for natural resistance are seldom investigated, although
`sometimes they can be deduced from mode of action studies.
`
`The ‘fungicide resistance’ we are considering here is a different phenomenon,
`sometimes called ‘acquired resistance’. Sooner or later during the years of commercial
`use of a fungicide, populations of the target pathogen can arise that are no longer
`sufficiently sensitive to be controlled adequately. They generally appear as a response
`to repeated use of the fungicide, or to repeated use of another fungicide which is
`related to it chemically and/or biochemically through a common mechanism of
`antifungal action. This emergence of resistant populations of target organisms, which
`were formerly well controlled, has been widely known for antibacterial drugs (e.g.
`sulphonamides, penicillin, streptomycin) and for agricultural and public health
`insecticides (e.g. DDT) for almost sixty years.
`
`Some people prefer to call this phenomenon ‘insensitivity’ or ‘tolerance’. The former
`term is preferred by some plant pathologists, because they believe that fungicide
`resistance is easily confused with host-plant resistance to certain species or pathotypes
`of fungi. Some agrochemical companies have also tended to use ‘insensitivity’, ‘loss
`of sensitivity’ or ‘tolerance’, because these sound less alarming than ‘resistance’. On
`the other hand, two studies on terminology recommended that ‘resistance’ should be
`the preferred term (Anon, 1979; Delp and Dekker, 1985). Also ‘resistance’ has been in
`use for many years to describe precisely the same phenomenon in bacteriology and
`entomology, and it is now very widely used with reference to fungicides also.
`
`Workers within the agrochemical industry have objected from time to time to the use
`of ‘resistance’ to describe shifts in fungicide sensitivity occurring either in non-crop
`situations such as the laboratory or experimental glasshouse, or in the field but to a
`degree which is too small to affect disease control. They recommend that ‘resistance’
`should denote only situations where failure or diminution of crop disease control is
`known to have resulted from a change in sensitivity. It is true that observations of
`‘resistance’ generated in the laboratory, and detection of rare or weakly resistant
`
`8
`
`

`

`FUNGICIDE RESISTANCE IN CROP PATHOGENS:
`HOW CAN IT BE MANAGED?
`
`variants in the field, have on occasions been misinterpreted by scientific authors, or by
`commercial competitors, as indicating actual or impending failure of a product to
`perform in practice, when in fact good control was still secured.
`
`However, attempts to restrict in this way the meaning of such a broadly used term as
`‘resistance’ are bound to fail and to create more confusion. It is better to qualify the
`term when necessary. ‘Field resistance’ (in contrast to ‘laboratory resistance’) has been
`used sometimes to denote specifically a crop disease control problem caused by
`resistance. However, detection of some signs of resistance in the field can still be a far
`cry from having a control failure. It seems preferable to use ‘field resistance’ to
`indicate merely the presence of resistant variants in field populations (at whatever
`frequency or severity), and ‘practical resistance’ to indicate consequent, observable
`loss of disease control, whenever such precise terminology is necessary. ‘Laboratory
`resistance’ or ‘artificially induced resistance’ also are useful, precise terms which are
`self-explanatory. Some authors have claimed to find ‘field resistance’ in studies where
`the resistant variants actually were detected only after the field samples were subjected
`to subsequent selection by exposure to the fungicide in the laboratory. This is a
`borderline case, which is hard to categorise.
`
`OCCURRENCE OF RESISTANCE
`
`Table 1 gives a much condensed history of the occurrence of practical fungicide
`resistance world-wide, and lists major fungicide groups for which resistance is well
`documented. Leading examples are given of the more important diseases affected, and
`a few key literature references are cited. Up to 1970 there were a few sporadic cases of
`fungicide resistance, which had occurred many years after the fungicide concerned
`was introduced. With the introduction of the systemic fungicides, the incidence of
`resistance increased greatly, and the time taken for resistance to emerge was often
`relatively short, sometimes within two years of first commercial introduction. Many of
`the fungicides introduced since the late 1960s have been seriously affected, with the
`notable exceptions of the amine fungicides (‘morpholines’), fosetyl-aluminium,
`anilinopyrimidines, phenylpyrroles and some of the fungicides used to control rice
`blast disease (e.g. probenazole, isoprothiolane and tricyclazole), which have retained
`effectiveness over many years of widespread use. Some recently introduced fungicides
`
`9
`
`

`

`FUNGICIDE RESISTANCE IN CROP PATHOGENS:
`HOW CAN IT BE MANAGED?
`
`Table 1
`Occurrence of Practical Fungicide Resistance in Crops
`
`Date first
`observed
`(approx.)
`
`Fungicide or
`fungicide
`class
`
`Years of commercial
`use before resistance
`observed (approx.)
`
`Main crop
`diseases and
`pathogens affected
`
`Ref*
`
`1960
`
`1964
`
`1969
`
`1970
`1971
`
`1971
`
`1976
`
`1977
`
`1980
`
`1982
`
`1982
`
`Aromatic
`hydrocarbons
`Organo-mercurials
`
`Dodine
`
`Benzimidazoles
`2-Amino-pyrimidines
`
`Kasugamycin
`
`Phosphorothiolates
`
`Triphenyltins
`
`Phenylamides
`
`Dicarboximides
`
`Sterol Demethylation
`inhibitors (DMIs)
`
`1985
`
`Carboxanilides
`
`1998
`
`2002
`
`Quinone outside
`Inhibitors (QoIs;
`Strobilurins)
`Melanin Biosynthesis
`Inhibitors (Dehydratase) (MBI-D)
`
`20
`
`40
`
`10
`
`2
`2
`
`6
`
`9
`
`13
`
`2
`
`5
`
`7
`
`15
`
`2
`
`2
`
`Citrus storage rots,
`Penicillium spp.
`Cereal leaf spot and stripe,
`Pyrenophora spp.
`Apple scab,
`Venturia inaequalis
`Many target pathogens,
`Cucumber and barley,
`powdery mildews
`Sphaerotheca fuliginea
`& Blumeria graminis
`Rice blast,
`Magnaporthe grisea
`Rice blast,
`Magnaporthe grisea
`Sugar beet leaf spot,
`Cercospora betae
`Potato blight and
`grape downy mildew,
`Phytophthora infestans
`& Plasmopara viticola
`Grape grey mould,
`Botrytis cinerea
`Cucurbit and barley
`powdery mildews,
`S. fuliginea
`& Blumeria graminis
`Barley loose smut,
`Ustilago nuda
`Many target diseases
`and pathogens
`
`Rice blast,
`Magnaporthe grisea
`
`1
`
`2
`
`3
`
`4
`5
`
`6
`
`6
`
`7
`
`8
`
`9
`
`10
`
`11
`
`12
`
`13
`
`*References: 1. Eckert, 1982; 2. Noble et al. 1966; 3. Gilpatrick, 1982; 4. Smith, 1988; 5. Brent, 1982; 6. Kato, 1988; 7 Giannopolitis, 1978; 8
`Staub, 1994; 9. Lorenz, 1988; 10. De Waard, 1994: 11. Locke, 1986; 12. Heaney et al. 2000; 13. Kaku et al. 2003.
`
`10
`
`

`

`FUNGICIDE RESISTANCE IN CROP PATHOGENS:
`HOW CAN IT BE MANAGED?
`
`such as benzamides and carboxylic acid amides have not yet encountered serious
`resistance problems, possibly because of the management precautions which have
`been taken. Most of the older materials such as copper fungicides, sulphur,
`dithiocarbamates (e.g. mancozeb), phthalimides (e.g. captan) and chlorothalonil, have
`retained their full effectiveness in all their uses, despite their extensive and sometimes
`exclusive use over many years.
`
`Often the onset of resistance has been associated with total, or almost total, failure of
`disease control. Indeed it was growers’ observations of obvious and sudden loss of
`effect that generally gave the first indication of resistance. Of course it was necessary
`to show that resistance really was the cause, by checking for abnormally low
`sensitivity of the pathogen in tests under controlled conditions. There was, and to
`some extent still is, a temptation for growers and advisers to blame resistance for all
`cases of difficulty of disease control. There are many other possible reasons, such as
`poor application, deteriorated product, misidentification of the pathogen, unusually
`heavy disease pressure. However, there remained many examples where no other
`explanation was found, and where serious loss of control was clearly correlated with
`greatly decreased sensitivity of the pathogen population as revealed in laboratory tests
`on representative samples.
`
`Resistance of the kind just described, characterised by a sudden and marked loss of
`effectiveness, and by the presence of clearcut sensitive and resistant pathogen
`populations with widely differing responses, is variously referred to as ‘qualitative’,
`‘single-step’, ‘discrete’, ‘disruptive’ or ‘discontinuous’ resistance (Fig.1). Once
`developed, it tends to be stable. If the fungicide concerned is withdrawn or used much
`less, pathogen populations can remain resistant for many years; a well-documented
`example is the sustained resistance of Cercospora betae, the cause of sugar-beet
`leafspot, to benzimidazole fungicides in Greece (Dovas et al., 1976). A gradual
`recovery of sensitivity can sometimes occur, as in the resistance of Phytophthora
`infestans, the potato late blight pathogen, to phenylamide fungicides (Cooke et al.,
`2006). In such cases, resistance tends to return quickly if unrestricted use of the
`fungicide is resumed, but re-entry involving also a partner fungicide has proved useful
`in some instances.
`
`Sometimes, as in the case of the DMI fungicides, and of the 2-amino-pyrimidine
`fungicide ethirimol, resistance has developed less suddenly. In such cases, both a
`decline in disease control and a decrease in sensitivity of pathogen populations as
`
`11
`
`

`

`FUNGICIDE RESISTANCE IN CROP PATHOGENS:
`HOW CAN IT BE MANAGED?
`
`revealed by monitoring tests, manifest themselves gradually, and are partial and
`variable in degree. This type of resistance is referred to as ‘quantitative’, ‘multi-step’,
`‘continuous’, ‘directional’ or ‘progressive’ (Fig.1). It reverts rapidly to a more
`sensitive condition under circumstances where the fungicide concerned becomes less
`intensively used and alternative fungicides are applied against the same disease.
`
`The first appearance of resistance in a particular fungicide-pathogen combination in
`one region has almost always been accompanied, or soon followed, by parallel
`behaviour in other regions where the fungicide is applied at a similar intensity.
`Whether the fungicide also meets resistance in other of its target pathogens depends on
`the individual case. Generally it does occur in other target pathogens that have a
`comparable rate of multiplication, provided that the fungicide is used in an equally
`
`Low
`
`Resistance
`DISCRETE RESISTANCE
`
`High
`
`Low
`
`Resistance
`MULTI-STEP RESISTANCE
`
`High
`
`Frequency in population
`
`Frequency in population
`
`12
`
`Fig. 1
`Diagrams showing the
`bimodal and unimodal
`distributions of degree
`of sensitivity which are
`characteristic of the
`discrete and multi-step
`patterns of resistance
`development. Blue
`shading indicates
`original sensitive
`population, and red
`shading subsequent
`resistant population.
`
`

`

`FUNGICIDE RESISTANCE IN CROP PATHOGENS:
`HOW CAN IT BE MANAGED?
`
`intensive way. It is notable that rust fungi, despite their abundant sporulation and rapid
`spread, appear to be low-risk, seldom producing resistance problems (Grasso et al.,
`2006).
`
`Pathogen populations that develop resistance to one fungicide automatically and
`simultaneously become resistant to those other fungicides that are affected by the
`same gene mutation and the same resistance mechanism. Generally these have proved
`to be fungicides that bear an obvious chemical relationship to the first fungicide, or
`which have a similar mechanism of fungitoxicity. This is the phenomenon known as
`‘cross-resistance’. For example, pathogen strains that resist benomyl are almost
`always highly resistant to other benzimidazole fungicides such as carbendazim,
`thiophanate-methyl or thiabendazole. Sometimes cross-resistance is partial, even when
`allowance is made for the greater inherent activity of different members of a fungicide
`group.
`
`There is a converse phenomenon, ‘negative cross-resistance’, in which a change to
`resistance to one fungicide automatically confers a change to sensitivity to another.
`This is much rarer, but several cases are well characterised; one, involving
`carbendazim and diethofencarb, has been of practical importance and is discussed
`later.
`
`Some pathogen strains are found to have developed separate mechanisms of resistance
`to two or more unrelated fungicides. These arise from independent mutations that are
`selected by exposure to each of the fungicides concerned. This phenomenon is totally
`different from cross-resistance in its origin and mechanism, and is usually termed
`‘multiple resistance’. An example is the common occurrence of strains of Botrytis
`cinerea that have become resistant to both benzimidazole and dicarboximide
`fungicides.
`
`ORIGINS OF RESISTANCE
`
`Once it arises, resistance is heritable. It results from one or more changes in the
`genetic constitution of the pathogen population. There is overwhelming circumstantial
`evidence that a mutant gene that causes production of a particular resistance
`mechanism pre-exists in minute amounts in the population. Before the fungicide was
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`FUNGICIDE RESISTANCE IN CROP PATHOGENS:
`HOW CAN IT BE MANAGED?
`
`ever used in the field, such a mutation would confer no advantage to the growth or
`survival of the organism, and could well cause a slight disadvantage. Hence it would
`remain at a very low frequency, probably dying out and re-appearing spontaneously
`many times.
`
`Spontaneous mutations of all kinds are continually occurring in all living organisms.
`The rate of mutation can be increased greatly in the laboratory by exposing the
`organism to ultra-violet light or chemical mutagenic agents, and thus resistant mutants
`can be produced artificially. However, it cannot be assumed that such artificial mutants
`are necessarily identical in resistance mechanism or in other respects to those that arise
`in the field.
`
`Typically, a resistant mutant might exist at an initial frequency of the order of 1 in
`1000 million spores or other propagules of the pathogen. Amongst the survivors of a
`fungicide treatment, however, the resistant forms will be in much higher proportion
`(‘the survival of the fittest’). It is only when this reaches say 1 in 100 or even 1 in 10
`in the population that difficulty of disease control and the presence of resistant
`individuals will have become readily detectable. Thus the obvious onset of resistance
`is often sudden, but prior to this the resistance will have been building up insidiously
`at undetectable levels. If a fungicide treatment is very effective, with few survivors,
`selection will be very rapid. If the fungicide is only 80% effective, then after each
`treatment the number of variants will be concentrated only 5-fold and the build-up will
`be slower.
`
`Several fairly obvious but important deductions, which can influence assessment of
`risk and design of avoidance strategies, can be made from consideration of this simple
`process of mutation and selection. Accumulation of resistant mutants will be enhanced
`by higher frequency of treatment with the fungicide concerned, by a more effective
`application method or dose, by the presence of larger pathogen populations before
`treatment, and by greater spore production and shorter generation times in the
`pathogen.
`
`The selection process outlined above is based on much genetic analysis of sensitive
`and resistant strains, and on much field experience. However, it represents the simplest
`form of resistance, the discrete pattern referred to earlier, which is also termed ‘major
`gene’ resistance. One point mutation causing a single amino acid change in the target
`protein is responsible for a high level of resistance, and the sensitive and resistant
`forms fall into very distinct classes. This pattern is characteristic of resistance to
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`several major groups of fungicides including benzimidazoles, phenylamides,
`dicarboximides and QoIs. Other mutations in the target protein may give rise to lower
`levels of
`resistance. For example,
`the F129L mutation
`in
`the
`b-cytochrome target of QoIs causes only low levels of resistance in many pathogens,
`and hence is of little practical importance, in contrast to the G143A mutation which
`causes a high degree of resistance, and consequent loss of disease control (Gisi et al.
`2002).
`
`A somewhat different ‘polygenic’ process of genetic change is thought to underlie the
`‘quantitative’ or ‘multi-step’ pattern of resistance. Again resistance results from the
`selection of mutants, but in this case a number of different genes, each with a partial
`effect, appear to be involved. The more genes that mutate to resistance-causing forms,
`the greater the degree of resistance. This would account for the gradual observable
`development of resistance, and for the continuous range of sensitivity that can be
`found (Fig.1). Although the theory of polygenic resistance is widely accepted, it must
`be said that the genetic evidence for polygenic resistance in field isolates is rather thin.
`The best known and most studied examples of continuous resistance in practice have
`been in cereal powdery mildews, which are rather hard to study genetically, and some
`of the data are conflicting (Hollomon, 1981; Hollomon et al., 1984; Brown et al.,
`1992). Biochemical evidence for polygenic resistance to azole (DMI) fungicides
`indicates involvement of at least four resistance mechanisms which are discussed
`below. However, Sanglard et al. (1998) studying the human pathogen Candida
`albicans, found that different mutations in the same target-site gene may accumulate
`in a single strain, and their individual effects may be additive, or possibly synergistic.
`In this way polyallelic changes may contribute to multistep development of resistance.
`
`QoIs (strobilurins) are the first fungicide class to target a protein (cytochrome bc-1)
`that is encoded by a mitochondrial gene. DNA repair mechanisms are less effective for
`mitochondrial DNA than for nuclear DNA, and consequently mitochondrially encoded
`genes are more liable to mutation. The frequency of DNA base changes in
`mitochondrial DNA is further increased by its close proximity to reactive oxygen
`species generated during respiration. Depending on the impact of these mutations on
`fitness, resistance seems likely to develop quickly where target sites are encoded by
`mitochondrial genes. Onset of resistance to QoIs was in fact rapid in a number of
`pathogens, although it must be noted that benzimidazole resistance, resulting from a
`nuclear mutation, developed equally quickly.
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`FUNGICIDE RESISTANCE IN CROP PATHOGENS:
`HOW CAN IT BE MANAGED?
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`RESISTANCE MECHANISMS
`
`A large amount of experimental effort has focussed on this subject, particularly in
`academic laboratories. A broad outline of current information is give