FUNGICIDE RESISTANCE IN PLANT PATHOGENS Introduction: Fungicide treatments are a key component in the integrated management of many plant

diseases. The appearance of resistance has become an important factor in limiting the efficacy and useful lifetime of fungicides developed at increasingly higher costs. Fungicide resistance can arise rapidly and completely so that disease control is totally lost or it can be a more gradual process resulting in partial loss of control. Fungicide resistant plant pathogens are not new. Although the first confirmation of fungicide resistance was in 1960, there were few subsequent incidences up until 1970. Since then there have been more incidences, especially with the introduction of systemic fungicides. Also of concern has been the shortening of time taken for resistance to emerge, sometimes within two years of a new commercial fungicide introduction. Natural or inherent resistance is a 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. The fungicide resistance is actually refers to acquired resistance to fungicides. Sooner or later during the years of 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 related to it chemically and/or by biochemical mechanism of antifungal action. According to FRAC resistance should denote only situations where failure or diminution of crop disease control is known to have resulted from a change in sensitivity (Brent and Hollomon, 2007). Pathogen resistance to fungicides is widespread. The performance of most of the modern, systemic fungicides that operate against single biochemical targets has been affected to some degree. Resistance development: A mutant gene that causes production of a particular resistance mechanism pre-exists in minute amounts in a population. Since such a mutation would confer no advantage to the growth or survival of the organism, it would remain at a very low frequency. Resistance builds up through the survival and spread of these rare mutants, during exposure to fungicide treatment. This development can be discrete (resulting from a single gene mutation) or gradual (considered to be polygenic). Random mutations resulting in biochemical resistance are passed along to the next generation. Repeated fungicide applications select for these mutants in a population. When the frequency in the population is high enough practical resistance is obtained. Patterns of resistance development There are two kinds of resistance phenotypes Qualitative

- Quantitative Qualitative resistance development Resistance of this kind 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. 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 welldocumented example is the sustained resistance of Cercospora betae, the cause of sugar-beet leaf spot, to benzimidazole fungicides. This type of resistance development seen in the case of high resistance risk fungicides would be typical for MBC, QoI and phenylamide resistance. Individuals are immune to the fungicide group. Quantitative resistance development Decline in disease control and a decrease in sensitivity of pathogen populations as 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. 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. This type of resistance development seen in the case of moderate resistance risk fungicides would be typical for DMI and dicarboximide resistance. Individuals are tolerant to the fungicide group. Table 2. Comparison of qualitative and quantitative resistance Qualitative resistance Quantitative resistance single-step, discrete, disruptive or multi-step, continuous, directional, discontinuous resistance shifting or progressive resistance Stable Unstable Monogenic Polygenic Immune to fungicide Tolerance to fungicide Sudden and marked loss of effectiveness Gradual with variable degree of loss of effectiveness E.g. MBC, QoI and phenylamide resistance E. g. DMI and dicarboximide resistance Mechanisms of Fungicide Resistance Resistance mechanisms vary, but mainly involve modification of the primary site of action of the fungicide within the fungal pathogen. Resistance may emerge as a result of genetic change and also due to physiological and biochemical changes in the fungal cell () Genetic mechanisms of fungicide resistance 1. Mutational changes Single gene mutations and multiple gene mutations leads to resistance development through biochemical and physiological alterations. Single gene mutation shows a rapid shift

towards resistance leading to a population which is predominantly resistant and fungicide is no longer effective in controlling the disease. Table 3. Examples of single gene mutations: Fungicidal group Position of mutation Change in aminoacid Benzimidazoles Codon 198 of tubulin gene Glutamic acid to alanine Dicarboximides Position 35 of histidine kinase gene Isoleucine to serine Strobilurins Position 143 of Cytochrome b gene Glysine to alanine 2. Gene interaction Interallelic and intraallelic gene interaction occurs if more than one locus mutates to impart fungicide resistance. When multiple genes are involved shift to the development of resistance is gradual since mutation should occur at all genes simultaneously which are very rare. It usually gives rise to more complex resistance mechanisms involving many loci. 3. Chromosomal control Fungicide resistance is controlled by chromosomal genes. No extra chromosomal genes are usually involved. 4. Phenotypic adaptation As a result of mutation the fungus may adjust its phenotype so as to survive under unfavourable conditions, resulting in the development of resistant strains of the pathogen. Biochemical and physiological mechanisms of fungicide resistance 1. Modification of the target site/ Alternate metabolic pathways The commonest mechanism appears to be an alteration to the biochemical target site of the fungicide. This could explain why many of the older products have not encountered resistance problems. Once they have penetrated the fungal cell, the older fungicides act as general enzyme inhibitors, affecting many target sites (hence they are sometimes called multisite inhibitors). Many sites in the fungus would have to change simultaneously in order to stop the fungicide from working. Genetically the chances of this happening are negligible, and in any case an organism with so many alterations would be highly unlikely to be pathogenic or even viable. Most of the more modern fungicides appear to act primarily at single target sites, and are often referred to as single-site or site-specific fungicides. Thus just a single gene mutation can cause the target site to alter, so as to become much less vulnerable to the fungicide. Substitution of glycine by alanine at position 143 of cytochrome b gene was found in resistant isolates against QoI fungicides. Decreased affinity to MBC by modification in tubulin target site imparts resistance to carbendazim and thiabendazole in Aspergillus nidulans. Circumvention or alternate metabolic pathway is also seen in resistant isolates. In QoI resistant Mycosphaerella graminicola isolates electron transport bypasses through alternate oxidase pathway and overcome the fungicide toxicity. There could be compensation of target site through overexpression of the target site. Overexpression of CYP51 gene causes DMI resistance in Blumeriella jaapii, Venturia inaequalis and Penicillium digitatum. Also 6- azauracil resistance in Cladosporium cucumerinum is brought about by the same mechanism.

2. Reduction in uptake and exclusion of the toxicant Resistance to imazalil and fenarimol is based on reduced uptake of fungicide by mycelium of the resistant mutant. Example: Aspergillus nidulans. Efflux systems have evolved in fungi to confer resistance against naturally occurring toxic substances, e.g. phytoalexins or phytoanticipins formed by plants as a response to pathogen attack. It is thus not surprising that these transporters also confer fungicide resistance. Two families of plasma membrane-localized efflux transporters are known to be involved in secretion of toxicants, i.e. ATP-Binding Cassette (ABC) transporters and the Major Facilitator Superfamily (MFS) transporters. ABC transporters, encoded by the AtrB gene involved in resistance to QoI detected in Aspergillus nidulans. Overexpression of ABC transporters confered DMI resistance in Botrytis cinerea and Mycosphaerella graminicola. 3. Detoxification of the toxicant The fungicide or its fungitoxic moiety is converted to non-toxic product by the resistant strains. Botrytis cinerea resistant isolate could make more nonvital thiol compound available for detoxifying captan. Dodine resistant strains of Nectria haematococca f.sp. cucurbitae also shows detoxification of compound. Resistance to organic mercury fungicides by Drechslera avenae is also brought about by the same mechanism. Lack of conversion of fungicide to toxic form is absent in the case of some fungicides which need to be converted to toxic form for fungitoxicity. Conversion of fungicide pyrazophos to 2 hydroxy- 5- methyl -6- ethoxycarbonylpyrazolo(1,5 - a) pyramidine (PP) is absent in resistant strains of Pyricularia oryzae.