Mode of action

Fungicides inhibit fungal growth by interfering with critical cellular processes.  Mode of action (MOA) refers to the specific cellular process inhibited by a particular fungicide.  FRAC currently lists 11 modes of action in its Mode of Action Poster and Code List (Table 1, column 1).  Fungicides may have a mode action that is not fully understood at the time of introduction.  Until there is specific evidence of the involved biochemical processes, FRAC list these compounds as “Unknown Mode of Action” in the FRAC code list. 

Within each mode of action there are specific sites of action.  These sites of action or target sites are the specific enzymes in a cellular process to which the fungicides are binding.   For example, both strobilurin fungicides and SDHI fungicides share the same MOA (inhibition of respiration) but have different sites of action in the respiratory pathway; SDHI’s inhibit complex II while strobilurins inhibit complex III.   In pharmaceutical literature the inhibition of a specific enzyme is referred to as the mechanism of action (some pathologists use the term mechanism of action interchangeably with mode of action).  FRAC assigns compounds active at the same target site a number (e.g. the SDHIs are FRAC group  #7 on the FRAC Code list). 

To understand the biochemical interaction of the fungicide with a specific target site, the analogy of using a lock and key to open a door is useful.  The lock is the target site on the enzyme and the key is the natural substrate the enzyme interacts with to complete normal cellular processes (analogous to unlocking the door). The fungicide(s) active at that target site are an additional set of key-like objects which can also fit into the lock.  If one of these “artificial” keys is in the lock, the normal key/substrate cannot fit into the lock and the fungal biochemical process is blocked (the door cannot be unlocked). Inside the cell, the fungicide and the substrate compete for the lock/target site.  As the fungicide accumulates in the cell, the normal substrate can no longer compete for the target site and normal cellular processes will reach such a low level, or may be blocked entirely, that adverse effects are observed.  While the fungicides share similarity with the fungal substrate in terms of their three-dimensional structure, they are not identical.  It is possible that alterations to the lock/target site could occur that will allow the fungal substrate to continue to bind and normal cellular processes to proceed, but not allow the fungicide(s) to bind (i.e. the lock could be changed such the original key still works to unlock the door but the key-like fungicide no longer fits in the lock).   This situation results in one specific type of resistance known as target-site resistance.

Fungicides active at the same target site (i.e. that is within the same FRAC code # on the FRAC Code List) are generally considered to be cross-resistant to each other.  Cross-resistance is a phenomenon that occurs when resistance arises to one fungicide that also results in resistance to another fungicide.  Occasionally, cross-resistance can occur between compounds active at different target sites (see multi-drug resistance under mechanisms of resistance below). The actual target site is not completely understood for some fungicides so the target site description remains rather generic.  For example, the target site description of the azanapthalenes is signal transduction.  The two azanapthalenes, quinoyxfen and proquinazid, are grouped together in the same FRAC group since cross resistance was observed in Erysiphe necator.  Interestingly, no cross-resistance was observed in another powdery mildew species, Blumeria graminis  (Genet and Jaworska, 2009).  Negative cross-resistance can also occur.  Negative cross resistance is when a change results in a reduction in sensitivity to one fungicide and an increase in sensitivity to another fungicide.  For example, isolates of Botrytis cinerea with reduced sensitivity to the benzimidazole fungicides (FRAC group #1 on the FRAC code list) have an increased sensitivity to the N-phenyl carbamates (FRAC group #10 on the FRAC code list; Leroux et al 1989).

Mechanisms of Resistance

There are 4 main mechanisms by which fungi can become resistant to fungicides.
  1. Alteration of the target site so that sensitivity to the fungicide is reduced:  By far the most common way that fungi can become resistant to a specific fungicide is via a change at the target site. As fungi grow their DNA is replicated when new cells are created.  This process of replication is imperfect and errors can occur.  These errors are known as mutations.  Since DNA is the code used to produce enzymes in the cell, some mutations result in changes to the amino acid sequence of the target site which in turn alters the shape of the lock/target site.  The fungicide/key may not fit as well anymore or may not fit at all in the target site/lock.  This results in a reduction in sensitivity that may run the gamut from small to very large.

  2. Detoxification of the fungicide:  The fungal cell contains a vast array of metabolic machinery for normal cellular processes.  This metabolic  machinery may be able to  modify the fungicide to a non-toxic form  that is no longer harmful to the cell.  Some fungicides are applied as inactive pro-fungicides which require further metabolism by the fungal cell to become the active form.  If fungal metabolism is altered such that the activation step does not occur the active form of the fungicide is not produced. 

  3. Overexpression of the target:  As discussed above, the fungicide is “competing” with the natural substrate for the target site.  As more and more fungicide enters the cell, it out-competes the natural substrate for the target and shuts down critical cellular processes as a result.  The production of additional target site enzyme (i.e. overexpression of the target) may increase the likelihood that enough of the fungal substrate will be able to bind with the target site enzyme such that cellular processes such as respiration can occur to some degree.  Higher doses of the fungicide in in vitro experiments may again restore the balance in the favor of the fungicide, but higher doses may not always be practical under field conditions.

  4. Exclusion or expulsion from the target site: Efflux pumps exist naturally within the cell to exclude or expel foreign substances or to export endogenous substances.  In fungi, the most common efflux pumps are ABC and MFS transporters.   Despite these efflux pumps most fungicides can reach effective concentrations inside the cell and inhibit cellular processes.  Occasionally, these transporters are successful in expelling enough of the fungicide such that the isolate has reduced sensitivity. The fungicides expelled from the cell by a specific transporter may or may not be active at the same target site; i.e. there is not a direct relationship between the transporter that expels a specific fungicide and the target site of the fungicide.  Multidrug resistance (MDR) develops when a specific transporter is able to exclude multiple fungicides from different target site groups.  Application of the fungicides in question may exert enough selection pressure that isolates containing these fungicide-exporting transporters become more prevalent in the population as is the case in Botrytis cinerea (Kretschmer et al. 2009).

References

Genet JL, Jaworska G.  Baseline sensitivity to proquinazid in Blumeria graminis f. sp. tritici and Erysiphe necator and cross‐resistance with other fungicides. Pest management science 2009; 65(8): 878-884.

Leroux P, Gredt, M. Negative cross-resistance of benzimidazole-resistant strains of Botrytis cinerea, Fusarium nivale and Pseudocercosporella herpotrichoides to various pesticides.  Netherlands Journal of Plant Pathology 1989; 95(1): 121-127.

Kretschmer M, Leroch M, Mosbach A, Walker AS, Fillinger S, Mernke, D, Schoonbeek HJ, Pradier JM, Leroux P, De Waard M,  Hahn M. Fungicide-driven evolution and molecular basis of multidrug resistance in field populations of the grey mould fungus Botrytis cinerea. PLoS pathogens 20095(12);e1000696.

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