Fungi Resistance to Multissite Fungicides

Multisite fungicides have been used for many years in fruit and vegetable crops worldwide. Cases of the fungi resistance development to these fungicides have been rare. From the 2002 season onwards, with the outbreak of Asian soybean rust in Brazil, caused by Phakopsora pachyrhizi, site-specific fungicides became the main weapon for its control. From 2002 to 2011, penetrant mobile site-specific fungicides were used and until today in double (DMI + QoI) or triple (DMI + QoI + SDHI) co-formulatoons in an area of more than 30 million hectares and with three sprays per area. This resulted, as expected, in the fungus sensitivity reduction, today with cross and multiple resistance to those site-specific fungicides. From the 2011 season in an attempt to recover control that for some chemicals and mixtures reached < 30%, research was started with site-specific + multi-site mixtures, taking as example Phytophthora infestans resistance development to metalaxyl in Europe showinig long-lasting solution found by the addition of multisite mancozeb. It is expected that the effective life of site-specific + multi-site mixtures may be as long in controlling soybean rust as it has been for potato, tomato and grape downy mildews. This review presents the concepts involved in the sensitivity reduction to fungicides. Some fungal species and fungicides involved are listed. Considering the P. pachyrhizi sporulation potential, the great soybean area sprayed and the number of sprays per area mainly with site-specific co-formulations and the reduced area sprayed with multisites, we discuss the need for annual monitoring of P. pachyrhizi sensitivity to the these chemicals.

From the 1970s, the resistance of phytopathogenic fungi became a problem with the predominant use of mobile-penetrating fungicides that were site-specific (Klittich, 2008).
On the other hand, the resistance of fungi to multisite fungicides (arylaminopyridine, chloronitriles, dithiocarbamates, copper, tin and mercury derivatives, phthalimides, sulfur, etc.) is still a rare event. The difficulty is due to the low probability of the occurrence of the minimum necessary number of mutations at different loci in the same fungus. On the contrary, with the introduction and repeated use of site-specific, acquired resistance has become common but incomparable to multisite (van den Boch & Gilligan, 2008).

Fungicides
Fungicides are synthetic or natural chemical compounds, or biological organisms capable of killing or inhibiting fungi, or the germination of fungi and oomycete spores (Mueller et al., 2013).

Fungitoxicity
Fungitoxicity is the property that a chemical substance has of being toxic to fungi and stramenopylae (pseudo fungi or chromists) in low concentration. This property is a molecule attribute.

Mode of Action, Mechanism of Action or Biochemical Mechanism of Action
The chemical structure of the fungicde active ingredient (a.i.) defines its mode of action by determining its uptake, movement in the plant, and its ability to reach and bind to the site of action-the physical location where the fungicide acts (Delp & Dekker, 1985). Mode of action is the process by which a chemically active substance produces an effect on a living organism or on a biochemical system. Or, the mechanism refers to the biochemical interaction through which the substance produces its toxic effect (Hewtt, 1988;Latin, 2017;Mueller et al., 2013).

Site of Action
Site of action, or target site, are specific enzymes in cellular processes to which the fungicide binds (Hewitt, 1988).

Sensitivity (of Sensitive, That Feels)
Property of the fungus to receive changes from the environment and to react to them. Sensitivity is an attribute of the fungal species (Reis et al., 2019).

Insensitivity
Not all fungi are sensitive to all fungicides (spectrum of action); some are always insensitive to certain molecules. For example, fungi of the genera Alternaria, Bipolaris, Curvularia, Drechslera, Exserohilum are insensitive to benzimidazole fungicides; on the other hand, benzimidazoles are not fungitoxic to these genera. Another example is the insensitivity of oomycetes, which cause mildews, to triazoles and benzimidazoles (Reis et al., 2019).
A fungus sensitive to a fungicidal molecule may have altered sensitivity, which is why it is said to have developed resistance. However, an insensitive fungus will never become sensitive.

Control Failure
The resistance of plant pathogenic fungi to fungicides is observed as a control failure or as a reduction in the performance of the fungicide; in this situation, farmers often react by increasing the dose and/or by reducing the interval between sprayings. In the next step, field experiments confirm the control failure. Situation in which the farmer observes that, when compared to previous crops, the fungicide efficiency was reduced. He says that there was a "failure of control" and starts to complain and seeks explanations for the fact (Reis et al., 2019).

Loss of Sensitivity
The word loss implies total insensitivity, which is not always true. Nevertheless, the concept of loss can be delimited following the Edgingnton & Klew (Edgington & Kew, 1971) criterium. Thus, it can be considered as sensitivity loss, or non-toxic, when the fungus presents inhibitory concentration, IC 50 , > 50 mg/L to a fungicida, and when lower than 50%, sensitivity reduction.

Sensitivity Reduction
Reduction is a slow process, requiring the application of a site-specific fungicide for many seasons and over a large area such as P. pachyrhizi and the DMIs (FRAC group 3, demethylase inhibitors), QoIs (Group 11, quinone outside inhibitors) and SDHIs (Group 7, succinate dehydrogenase inhibitors) fungicides. The reduction is present when the inhibitory concentration (IC 50 ) increase over time for the mycelial growth, spore germination or disease control. Therefore, in most cases, what is happening is a slow reduction instead of sensitivy loss.
Molecular techniques are useful in proving the presence of reduced sensitivity after resistance has been quantified in laboratory bioassays (Hollomon, 2015).

Erosion of the Fungicide
Expression taken as a synonym for reduced sensitivity of a fungus to a given fungicide (Hahn, 2014).

Resistance
Fungicide resistance is the result of the adaptation of a fungus to a fungicide due to its stable hereditary genetic alteration leading to the emergence and spread of mutants with reduced sensitivity to the fungicide (Delp & Dekker, 1985). The term proposed by FRAC (2019) refers to a stable and hereditary adjustment of a fungus to a fungicide, resulting in a reduction in the pathogen sensitivity. This adjustment results in a 'considerable' reduction in the sensitivity of the pathogen to the chemical compound, which can be partial or total, always with an increase in the IC 50 [sensitivity reduction factor (SRF)] > 1.0. This ability is gained through evolutionary processes (Mueller el al., 2013).

Cross-Resistance
Fungicides of the same chemical group, for example tebuconazole and cyproconazole, have different chemical structures. However, both have the same toxicity to fungi. Therefore, both are considered demethylation inhibitor fungicides (DMI), a name that expresses the same-shared mode of action. This fact means that even if you rotate two within the same fungicide group, the fungus detects them as being the same fungicide. It also means that if resistance develops for one member of the group, it will be present for all other members of that family. The resistance is called crossed reaching all group members (EPPO, 1998).

Multiple Drug Resistance (MDR)
It is the sensitivity reduction to various fungicides with different modes of action shown by a fungus specie. MDR is defined as the acquired sensitivity reduction of at least one fungus to at least three fungicides with a distinct mechanism of action. The main resistance mechanism involved here is the overexpression of the efflux transporter genes present in the plasma membrane. It results in increased cellular expulsion of the fungicide reducing the fungus sensitivity to several unrelated fungicides (Alekshun & Levy, 2007;Chapman et al., 2011;Chen et al., 2017;Hahn & Leroch, 2015;Leroux et al., 2002).

Acquired Resistance
It refers to a fungus that in the wild state was sensitive to the fungicide and that developed resistance after exposure to the chemical (Hollomon, 2015). This is what happens with fungicides applied to control diseases in the field.

Site-Specific, Monosite or Unisite Fungicide
Of the millions of biochemical reactions that take place in the fungus cell, the site-specific fungicide (monogenic resistance) interferes with only one biochemical site (an enzyme). This is a vital enzyme for the fngus physiology, so if it is blocked, the fungus will die. Fungicides with a site-specific mode of action are at high risk for the development of resistance compared to multiple-site fungicides (Mueller et al., 2013).

Multisite Fungicide
It refers to the fungicide that paralyzes at least five metabolic processes of the fungus (Mueller et al., 2013). For this reason, the development of resistance to them has not yet been frequently reported.

Site-Specific and Penetrant Mobile
Many use these two terms considering that all site-specific are penetrant mobile, however iprodione is a site-specific, signal transduction inhibitor, non-penetrating with protectant and some eradicant activity (PPDB, 2021).

Fungicide Effective Life
The effective life of a fungicide is the time from its introduction on the market for use in the control of a given fungus, until the moment when efficient control is no longer obtained due to the development of resistance of the target fungus (Hobbelen et al., 2011).

Mechanisms of Fungi Resistance to Fungicides
How do fungi defend theselves against fungicides? There are four main mechanisms by which fungi become resistant to fungicides.
For a better understanding of the mechanisms, the functions of cell organelles involved in the defense mechanisms of fungi are briefly reviewed.

Plasma Membrane
The cell, or cytoplasmic, membrane is a biological membrane that has selective permeability to organic molecules and ions. Controls the movement of substances into and out of the cell. This membrane is made up of a double layer of phospholipids and interspersed with proteins embedded in it. The membrane is said to be semi-permeable in that it can let a substance (molecule or ion) pass freely, pass through a limited form, or pass at all. Membranes also contain receptor proteins that allow cells to detect external signaling molecules such as hormones (Ishii & Hollomon, 2015).
The main mechanisms of fungi resistance to fungicides are: (a) Substance transport across the plasma membrane: To reach the intracellular organelles, the fungicide has to cross the plasma membrane with a complex constitution.
There are three forms of transport across the cell membrane (Alekshun & Levy, 2007;Ishii & Hollomon, 2015;Ward et al., 2006). The movement of substances across the membrane can be passive by simple diffusion, or by diffusion facilitated by the transport proteins channel following a positive concentration gradient and active, with energy consumption (ATP) against a concentration gradient.
(b) Change in target site reducing sensitivity to fungicide: The most common mechanism of resistance is a change in target site (enzyme) in the fungus and occurs only with site-specific fungicides, which dominated the market after 1970. Multisite fungicides, most of those developed since 1969, are not prone to the development of resistance at the target or action site. As the fungus grows, its DNA is replicated when new cells are created. This replication process is imperfect and errors can occur. Such errors are known as mutations. DNA is the code used to produce enzymes in the cell, and some mutations result in a change in the target site's amino acid sequence which in turn alters the shape of the receptor site (lock) of the fungicide. Thus the (key) fungicide may not fit into the site (lock) resulting in a partial or total reduction of the fungus' sensitivity to the fungicide. Therefore, an alteration by mutation in the fungicide target of action reduces the drug fitness through this target site, resulting in reduced sensitivity (Ishii & Hollomon, 2015).
(c) Gene overexpression: Gene overexpression is the abnormal production of large amounts of a substance which is encoded by one or more genes. In the case of overexpression, the target enzyme does not undergo any change (mutation). Instead, the pathogen produces it in large quantities (Cools et al., 2012).
For example the overexpression of the Cyp51 gene. Azole fungicides (DMIs) inhibit the Cyp51 gene encoding the demethylase enzyme involved in the ergosterol biosynthesis process. The fungus to defend itself from the effect of the fungicide increases the production of the enzyme in order to produce much more enzyme (demethylase) so that ergosterol is still produced even in the presence of the fungicide. Due to the increased production of the enzyme, the amount of fungicide present in the cell is not enough to couple with all the available enzyme, completely blocking the production of ergosterol. This leaves an amount of free enzyme without the coupling of the fungicide, producing enough of the ergosterol to keep the cell alive. In this case, the amount of fungicide is not enough to completely inhibit ergosterol production. Gene overexpression results in greater production of demethylase beyond normal. Therefore, even though triazole inhibits part of its synthesis, there is still an amount of enzyme remaining, maintaining the cell's functional activity (Alekshun & Levy, 2007;Hahn & Leroch, 2015;Leroux et al., 2001;Price et al., 2015;Ward et al., 2006).
(d) Exclusion of the fungicide from the cell: The cell's efflux is the elimination of a certain substance from its interior to the outside. Active efflux is a condition where pathogen cells pump the fungicide out of the cell faster than it accumulates to a toxic concentration. However, the target site remains unchanged. Active efflux prevents the accumulation of sufficient concentration to stop cell function and fungus growth.
Unlike influx, entry to the interior of the cell, efflux pumps occur naturally in cells that exclude or expel foreign substances or import substances useful to their metabolism. In fungi, the most common efflux pumps are protein transport pumps. Occasionally, these transporters succeed in expelling sufficient amounts of the fungicide from within the cell. Transport or carrier proteins in the plasma membrane are responsible for the active efflux of foreign material, including fungicides (Price et al., 2015).
There are two types of efflux: (i) Passive efflux is the expulsion of a certain substance to the outside of a cell (movement, or passive diffusion). (ii) There is also the participation of an efflux pump, which consists of the active pumping of the fungicide from the intracellular to the extracellular environment, that is, the active efflux. Efflux pumps are transmembrane proteins that can act to expel fungicides against a concentration gradient. There may also be an oveexpression of efflux pumps consisting of an increase in the concentration of their number (Hahn & Leroch, 2015). Multiple drug resistance is related to the overexpression of transport proteins.
(e) Detoxification or molecule inactivation by thiols overproduction: Substances that inactivate molecules of fungicides, has been suggested as the most likely mechanism that confers fungal resistance to mancozeb (Barak & Edgingtom, 1984;Gilpatrick, 1982;Yang et al., 2019). However, the mechanisms that confer resistance to this fungicide are questionable and very complex to be clarified. The main genomic and molecular study was carried out with the yeast Saccharomyces cerevisiae, having determined 286 genes that would be involved with resistance to a xenobiotic (Dias et al., 2009).
Many papers have been published on the fungi resistance to multisite fungicides, some were selected as an example (Table 1).

Final Remmarks
Although, fungal resistance to iprodione, a nonpenetrant fungicide, has been reported, it was not included in this review due to its site-specific mode of action.
The number of site-specific fungicide molecules marketed is considerably higher than multisite. From the 1970s onwards, site-specifics dominate the world market, being used to control diseases in a greater number of plant species, in a larger area and with a greater number of sprayings per season, in addition to their high risk to resistance development. This has resulted in the largest number of citations of site-specific resistance.
It is likely that for all commercialized, both multisite and site-specific fungicides, regardless of their active principle and resistance mechanism, at least one fungus resistant to them has already been reported. However, as site-specifics dominate the market, the large volume published focuses on this group.
Based on the consulted literature, even new site-specific mechanisms of action developed in the future will have the potential to select, in a few seasons, fungi resistant to them, shortening their effective life.
Although, Bordeaux mixture is considered the oldest foliage protectant fungicide, developed in 1885, no resistance of Phytophthora infestans (Mont.) de Bary to this fungicide was found in the consulted literature. Perhaps it is the fungicide with the longest effective life in the history of downy mildew chemical control on potatoes, tomatoes and grape. Are the cuprics the hardest to be defeated by fungi? In this sense, in the consulted literature only two reports of reduced sensitivity of fungi to cupric fungicides were found, but to a large number of species of phytopathogenic bacteria (Lamichane et al., 2018).
In the available literature, no reports were found on rust fungi resistant to mutissites.
It would also be important to determine the time required since it first use to the emergence of resistance or the duration of their effective life. According to the FRAC (2019), fungal resistance to pencycuron (phenylurea; recommended for the control of Rhizoctonia solani in the treatment of potato tubers) and to tricyclazole (triazolobenzothiazole) penetrant-mobile for the control of Pyricularia oryzae Cav in rice has not yet been reported.
Should the chemical industry continue to synthesize site-specific fungicides, as it has been doing intensively, even with a relatively short effective life as is happening with the new carboxamides towards Phakopsora pachyrhizi Sydow & Sydow.
In Brazil, the greatest use of multi-site fungicides (chlorothalonil, mancozeb and copper oxychloride) has been in soybean crop, to control P. pachyrhizi, the causal agent of Asian rust. Its use began in 2010/11, therefore beeing used in the last 10 seasons. To give an idea of the selection pressure that multi-sites are subject in soybean crop, in the 2020/21 season, the area cultivated with soybeans was > 38 million hectares, with 2.6 sprayings/ha, but with multi-site in an area of only 12%. What can happen with these multi-sites in the control of soybean rust under this situation? Should multissítes be used alone for ASR control?
At the moment, in Brazil, the use of multisite is the main weapon to face the development of P. pachyrhizi resistance to mobile penetrant site-specicif fungicides.
Multiple resistance is present in P. pachyrhizi to DMIs, QoIs and SDHIs and even so, these fungicides are applied in the largest area of soybean, without the multisite mixture, and thus, their efficacy has been reduced season after season. If the efficacy, which is already low, but has been reduced season after season, reaches < 30%, could the addition of multi-site revert the situation? Would multi-sites be used solo because they have superior control than site-specific?
Considering the chemical control of ASR in Brazil, with the well-defined presence of cross and multiple resistance to site-specific, reflected in a constant sensitivity reduction evolution of P. pachyrhizi, season after season, we will reach a situation in which the most efficient control would be achieved with multisites solo? Therefore, would multisites withstand the enormous selection pressure for resistance?
Let us remmember the development of P. infestans resistance to metalaxyl (in the 1977) and the solution given by the ready-made commercial mixture with mancozeb (in the 1980) would not be an indication that this would be the practice to be pursued in Brazil for economically sustainable control and fungicide with long effective life in controlling soybean rust? What has been the effective life of the metalaxyl + mancozeb mixture in controlling mildews and whether cases of mildew resistance to this mixture or similar ones has been reported? In the same direction, and similarly to ensure a long effective life in the control of P. pachyrhizi, the use of ready-made, liquid commercial mixture, containing IDM (prothioconazole and/or tebuconazole) and IQe (picoxystrobin and/or trifloxystrobin) + multisite (chlorothalonil), or mancozeb, or copper oxychloride) would be a solution?
The exposure time of P. pachyrhizi to multi-site fungicide is still too short to make a judgment about their effective life.