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The Botanical Review 69(2): 162172

The Role of Fungicides in the Physiology of Higher Plants:


Implications for Defense Responses
PABLO C. GARCA, ROSA M. RIVERO, JUAN M. RUIZ,

AND

LUIS ROMERO

Department of Plant Biology


Faculty of Sciences
University of Granada
18071 Granada, Spain

I. Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
II. Consequences of Pathogen Attack . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
A. Plant-Pathogen Interaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
B. Defense Responses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
III. Fungicides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
A. Use of Fungicides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
B. Types of Fungicides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
C. Action Mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
D. Phytotoxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
IV. Metabolic Processes Involved in Defense Responses of Plants: The Role
of Fungicides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
A. Bioactivity of Phenolic Compounds in Pathogen Resistance . . . . . . . . . . . . . . . .
B. Response of Oxidative Metabolism to Pathogen Attack . . . . . . . . . . . . . . . . . . . .
V. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
VI. Literature Cited . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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I. Abstract
Plants react to pathogen attack through a variety of active and passive defense mechanisms
primarily related to the metabolism of phenolic compounds and oxidative metabolism. Thus
the activation of defensive reactions is associated with the increased expression of a great
number of genes that encode enzymes involved in the biosynthetic pathway of phenolic compounds. Similarly, the activation of oxidative metabolism precedes the expression of defense
genes during plant-pathogen interactions, so both metabolic processes must exert a major function in directing the mechanisms to resist disease. Similarly, it has been suggested that certain
fungicides used to mitigate or prevent pathogen attack may be involved in activating certain
defensive responses of plants. However, the fact that such substances may influence the key
steps of the phenolic and oxidative processes has scarcely been studied. Our work confirms the
results proposed by other authors, who suggest that certain wide-spectrum fungicides, in addition to their antibiotic action against pathogens, may be involved in the activation of some
defensive responses of plants.

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Issued 16 December 2003


2003 The New York Botanical Garden

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II. Consequences of Pathogen Attack


A. PLANTPATHOGEN INTERACTION

The first step in the infection of a plant by a pathogen is penetration of vegetal tissue through
natural openings such as stomata or by injuries (Heydari & Misaghi, 1999). The invasion of the
tissue is often followed by swift development and spreading of the pathogen within the host. As
soon as the plant detects the presence of the invading microorganism, one or more defense
mechanisms can be triggered to restrict the growth of the pathogen and ultimately to destroy it
(Dixon et al., 1994; Benhamou, 1996). These defense mechanisms include both physical barriers and chemical barriers, such as reinforced cell walls or synthesis of antimicrobial molecules
(Hammond-Kosack & Jones, 1996).
Interactions between plants and pathogens have been classified as compatible or incompatible. That is, incompatible reactions (resistant host, avirulent pathogen) are often characterized
by the appearance of necrosis, or death of plant cells, at the point of pathogen penetration. Also
known as the hypersensitive response (HR), this prevents transmission of the pathogen to other
cells or parts of the host plant. On the other hand, in compatible interactions (sensitive host,
virulent pathogen), in which the pathogen can spread to other zones of the plant, massive plantcell death does not occur (Bowler et al., 1992).
B. DEFENSE RESPONSES

The HR involves a multitude of biochemical events in the plant, including: synthesis of


ethylene and phytoalexins; reinforcement of the cell wall with callose, lignin, and related compounds; accumulation of glycoproteins rich in hydroxyproline at the limit of the cell wall; and
synthesis of pathogenesis-related proteins (PRP), these including glucanases, quitanases,
proxidases, and protein inhibitors (Dixon & Lamb, 1990). During the development of HR the
spatial and temporal regulation of the different general defense reactions such as cell-wall
reinforcement, phytoalexin production, and antimicrobial protein accumulation count among
the most decisive factors governing susceptibility or resistance in plant-pathogen interactions
(Hammond-Kosack & Jones, 1996).
In addition to this genetic resistance, plants reveal an inducible resistance called systemic
acquired resistance (SAR), owing to necrotic lesions caused by pathogens in both the compatible interactions and the incompatible interactions (Chester, 1933; Hunt & Ryals, 1996; Ryals
et al., 1996). The development of SAR protects the plant against a broad spectrum of pathogens
and correlates with the induction of a group of well-characterized genes (SAR genes) that make
up the so-called PRP (Ward et al., 1991; Uknes et al., 1992). This response appears to be
modular, strengthening primary resistance mechanisms so that the SAR can convert a compatible interaction into an incompatible one (Uknes et al., 1992; Mauch-Mani & Slusarenko,
1996).

III. Fungicides
A. USE OF FUNGICIDES

Plant diseases are often treated with chemicals (e.g., fungicides, nematicides, bactericides)
and, in some cases, with cultivation techniques. Nevertheless, the use of chemicals to control
plant diseases has stirred public concern in recent years because of the harmful potential of
such substances both in the environment and in the food chain, as some chemicals have been
demonstrated to be carcinogenic. Other problems include the appearance of new strains of

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pathogens that have become resistant to existing chemical compounds, the gradual elimination
of some pesticides, and the refusal of some companies to develop new chemical products due
to the long registration process and associated costs (Heydari & Misaghi, 1999). In any case, to
maintain, and in some cases to increase, crop yield, farmers apply enormous quantities of
chemical treatments of all types each year, but, despite the heavy use of fungicides, an average
of 35% of the possible agricultural yield is lost to fungal attack (Bader & Abdel-Basset, 1999).
B. TYPES OF FUNGICIDES

The great variety of known fungicides can be classified in two main categories: contact and
systemic. Contact fungicides, such as copper (Cu) or sulfur (S) have preventive action by
killing the fungi as the spores germinate before the mycelia can grow and develop within the
plant tissues (Yuste & Gostincar, 1999). Systemic fungicides, also known as curative or eradication fungicides, can kill the fungus after the mycelia have penetrated the parenchyma of the
plant tissue, stopping the dispersal or infection within the plant (Yuste & Gostincar, 1999).
Benzimidazoles are a group of organic fungicides with systemic action that are extensively
used in agriculture. These types of compounds control a broad range of fungi at relatively low
application rates (Delp, 1987). In addition, it has been demonstrated that these compounds can
have beneficial effects on the physiology of the plant. For example, benomyl (carbamate of
methyl-N-butyl-carbamyl-benzimidazole), one of the most effective and extensively used benzimidazoles, has a cytokinin activity in soy and radish (Skene, 1972). Carbendazim (benzimidazole 2-il methyl carbamate), with properties similar to those of benomyl and of which it is an
active metabolite, delays senescence in wheat (Triticum aestivum L; Tripathi et al., 1982).
C. ACTION MECHANISMS

In general, most fungicides act by inhibiting the energy metabolism, blocking biosynthesis
or altering cell membranes of the fungus (Domnguez, 1998). Depending on where the cell is
attacked, fungicides can act on the cell wall, the cytoplasm membrane, the protoplast, the
mitochrondria, the ribosomes, or the nucleus. Thus, for example, carbendazim, a systemic
fungicide with curative and protective action, inhibits the development of the germinal tubes,
the formation of the apresoria, and the growth of the nucleus (Tomlin, 1994).
Although it is generally accepted that the activity of these compounds derives from their
direct antibiotic action on the invading microorganism, speculation has arisen concerning how
the plants defense systems may bolster the effectiveness of the fungicide (Ward et al., 1980).
Thus in soy, for example, the effectiveness of metalaxyl, a systemic fungicide active against
oomycete fungi (Fisher & Hayes, 1982), has been correlated with the accumulation of glyceolin,
an antimicrobial plant phytoalexin (Ward et al., 1980). Later it was reported that, in potato
tubers, metalaxyl is effective against Fusarium sp. and Alternaria sp., fungi for which metalaxyl
shows no direct toxicity (Barak et al., 1984). Similarly, it has been suggested that the fungicide
fosetyl and Cu (OH)2, wide-spectrum fungicides and bactericides, may be involved in activating some defensive responses in the host (Guest, 1984; Nemestothy & Guest, 1990; Jones et
al., 1991). In this respect, Molina et al. (1998) demonstrated that in Arabidopsis the innate
defense mechanism of the plant contributes to the effectiveness of various fungicides.
D. PHYTOTOXICITY

Apart from the simple idea that a biocide should be specific in its target (a fungicide should
be toxic for a fungus, an insecticide for an insect, etc.), only a few articles have been published

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on the issue of whether different chemical products alter or inhibit the physiological or metabolical activity of plants. In this sense, Bader and Abdel-Basset (1999) showed for the first time
that fungicides of the triforin type (saprol) strongly inhibit electron-transport reactions of chloroplasts. Benzimidazoles, though effective fungicides, can become phytotoxic, stunting plant
growth and causing visible damage to seedlings (Marc et al., 1996). Some products, such as
benlate DF (benomyl), depress photosynthesis (Marc et al., 1996). Finally, carbendazim does
not inhibit photosynthesis but does lower the levels of foliar calcium (Ca), promoting chlorosis
in Petunia (Marc et al., 1996). In some cases the cause of the phytotoxicity of the fungicide
proved to be related to certain products resulting from the breakdown of the fungicide itself
after penetration of the host cell and integration into the cell metabolism. Thus Marc et al.
(1997) demonstrated that dybutylurea, a product of the breakdown of benomyl, can be particularly responsible for the phytotoxicity of the fungicide benlate DF. Other ingredients of the
degradation of benlate also contribute to the phytotoxicity of the fungicide (Marc et al., 1997).
To understand the effects of fungicides on the physiology of plants, Garca et al. (2002)
analyzed the manner in which the different concentrations of a fungicide affect biomass production, the content of pigments, and the levels of nutrients by foliar application in tobacco
plants. The fungicide used in this experiment was carbendazim (carb) because it is one of the
most extensively used fungicides in southeastern Spain, a zone with intensive agriculture, and
because it is a broad-spectrum preventive fungicide, applied to a large number of crops (Tomlin,
1994). The fungicide was applied at 100% purity and at three different concentrations: 1.3 mM
(carb 1), 2.6 mM (carb 2), and 5.2 mM (carb 3). The control treatment consisted of not applying carbendazim. Taking into account all of the parameters analyzed in this experiment, these
researchers found that, in relation to control, the different treatments with carbendazim caused
generally similar effects. The application of the dosage lower than recommended (1.3 mM)
resulted in greater dry weight as well as in greater nitrogen (N) and potassium (K) concentrations than in control. This reflects the beneficial effect that the foliar application of carbendazim
can exert on the accumulation of these essential nutrients, as well as on the growth and development of tobacco plants. The application of the recommended dosage of carbendazim (2.6
mM) slightly reduced values, with respect to the lesser dosage, for the dry weight, nutritional
levels, all foliar pigments, P, Ca, and magnesium (Mg). These results reflect a harmful, though
not severe, effect of carbendazim on the nutritional staus, pigment biosynthesis, and biomass.
Therefore, the results, in revealing slight phytotoxicity, only partially support the recommendation of the use of this product. Finally, these authors found that the application of the dosage
heavier than recommended (5.2 mM) significantly reduced dry weight, all foliar pigments, and
nutrients with respect to the other dosages and control. These data clearly show that this fungicide, applied in excess, is toxic for healthy tobacco plants. Additionally, symptoms of necrosis
were found in old leaves after the application of increasing dosages of the fungicide.
In short, the experimental results imply that the negative effects of carbendazim can be
avoided by lowering the levels normally applied in agriculture. Nevertheless, the possibility
remains that this reduction in the application rate will result in lower effectiveness against
pathogens, although this problem requires further investigation.

IV. Metabolic Processes Involved in Defense Responses of Plants:


The Role of Fungicides
A. BIOACTIVITY OF PHENOLIC COMPOUNDS IN PATHOGEN RESISTANCE

Three groups of phenolic compounds are involved in plant defense against pathogens: lignins
and related polyphenolics; phenylpropanoids (phenolics and phytoalexins); and salicylic acid

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(Kuc, 1995; Wu et al., 1997; Wendehenne et al., 1998). On one hand, alkaline hydrolysis of the
cell walls of infected foliar tissue or potato tubers releases two types of phenolic compounds,
derivatives of hydroxybenzoic acid and amides of hydroxycinnamic acid (Friend, 1985). In
this respect, it appears that the incorporation into the cell wall of the derivatives of hydroxycinnamic acid (HCA), mainly cumaric, ferulic, and their conjugates, induced by pathogens,
should strengthen rigidity and diminish digestibility of the cell wall by the degrading enzymes of the pathogen (Nicholson & Hammerschmidt, 1992). In this way, phenolpropanoid
skeletons serve as substrates to begin the lignification process, which has recently been proposed as a mechanism of resistance to injuries provoked by fungal pathogens (Tiburzy &
Reisener, 1990).
On the other hand, it is known that in some plants phenolpropanoids are required for the
formation of phytoalexins (Dixon et al., 1994). Phytoalexins are defined as antimicrobial compounds synthesized in response to pathogen attack, since it has been confirmed that the levels
of these compounds increase sharply around the site of the infection until they reach concentrations toxic for the pathogens (Dixon et al., 1994). In this regard, many legumes, including soy,
green bean, and pea, respond to pathogen attack and other types of stress by accumulating
isoflavonoid phytoalexins (Ebel & Grisebach, 1988).
Apart from these compounds, it has been found that necrosis induced by pathogens in inoculated leaves is accompanied by the accumulation of salicylic acid (SA) at the infection site, as well
as in the phloem fluid and in healthy, uninoculated leaves (Malamy et al., 1990; Mtraux et al.,
1990; Enyedi et al., 1992; Summermatter et al., 1995). Additionally, SA is required to induce the
expression of a group of genes called SAR, a part of which encodes the PRP (Ward et al., 1991;
Delaney et al., 1994). For this reason, SA is believed to participate in the transduction process of
the signal that triggers SAR (Rasmussen et al., 1991; Delaney et al., 1994).
Finally, the accumulation of phenolic compounds in affected tissues is preceded by an increased coordination of activities of the principal enzymes involved in the phenolpropanoid
pathway. Thus the greater activity of polyphenol oxidase (PPO), guaiacol peroxidase (GPX),
and phenylalanine-ammonium liase (PAL) was noted in plants treated with various inducers of
resistance, both biotic and abiotic (Smith-Becker et al., 1998). Many works suggest that PAL is
the prime enzyme in regulating SA synthesis and in establishing SAR (Mauch-Mani &
Slusarenko, 1996). Thus plants respond to pathogen attack through a great variety of defense
mechanisms, in which the metabolism of phenolic compounds is decisively involved and therefore largely determines the consequences of the plant-pathogen interactions.
In addition to pathogen attack, different types of biotic and abiotic stress can induce phenolic metabolism, including injuries, ultraviolet rays, air pollution (especially ozone [O3]) and
thermal stress, with the effects of herbicides having been most extensively studied (Lydon &
Duke, 1989). Also, different works show that the status of certain nutrients, such as boron (B),
Ca, N, P, and iron (Fe) can prompt changes in phenolic metabolism.
However, the influence that can be exerted on this process by certain substances such as
fungicides, which are applied to plants specifically with the intention of mitigating and above
all preventing possible pathogen attack, has hardly been studied. Thus, in view of the essential
role of phenolic compounds in the development of resistance against pathogens and given the
influence that fungicides exert over plant diseases, Garca et al. (2001) determined the effect of
the application of different concentrations of carbendazim (1.3 mM, carb 1; 2.6 mM, carb 2;
5.2 mM, carb 3) on the metabolism of phenolic compounds in tobacco plants not suffering
biotic or abiotic damage. Their results indicated that the application of carb 1 increased PAL
activity as well as the accumulation of phenolics. Therefore, in the case of carbendazim, the
application of a fungicide concentration of 50% less than recommended (carb 1) may be more

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effective in fortifying plant resistance to pathogen infection, given that Nagarathna et al. (1993)
suggested that increased PAL activity and therefore greater phenolic accumulation increased
lignin synthesis, resulting in a hypersensitive response at the infection site and ultimately preventing development of the fungus.
On the other hand, Garca et al. (2001) found an inhibition of phenolic oxidation by the
application of the highest fungicide concentration, primarily with carb 3. These findings suggest that the excessive application of carbendazim (5.2 mM) may harm healthy tobacco plants,
since inhibited phenolic metabolism (biosynthesis and oxidation) would appreciably weaken
the plant response to pathogen attack. In this sense, it has been observed that tobacco plants in
which PAL activity had been epigenetically suppressed, thereby activating phenolic metabolism, were incapable of expressing SAR (Pallas et al., 1996).
B. RESPONSE OF OXIDATIVE METABOLISM TO PATHOGEN ATTACK

Oxidative metabolism in plants is mediated by reduced oxygen species such as oxygen (O2)
and hydrogen peroxide (H2O2) radicals, which are inevitable by-products of cell metabolism.
Separately, both species are not extremely active, but together they can form the radical hydroxyl anion (OH.) as well as other highly reactive derivatives. These free radicals can cause
lipid peroxidation, protein denaturalization, and genetic mutations (Cadenas, 1989). To prevent this oxidative damage, plant cells are equipped with a system of antioxidant molecules and
protective enzymes to detoxify these radicals, lessening damage to the cell metabolism.
Therefore, plant cells have the ability to degrade and detoxify so called activated oxygen species (AOS). Thus, under normal conditions, most cells have several mechanisms to maintain AOS
levels as low as possible within the cell. However, under certain stress conditions, such as pathogen
attack, these protective mechanisms are rerouted through a rapid and transitory production of large
quantities of AOS called oxidative burst (Doke, 1983). This event is one of the earliest visible
responses during the development of defense mechanisms in plant-pathogen interactions. Thus this
phenomenon has been observed in plants infected with pathogenic microorganisms, including fungi,
bacteria, viruses, and pathogen cell-wall fragments, or in response to mechanical stress (Doke &
Ohashi, 1988; Keppler et al., 1989; Vera-Estrella et al., 1992).
The main AOS of oxidative burst is H2O2, with the possible participation of O2., since it has
been demonstrated that this process is sensitive to the addition of catalase (CAT) to the medium
and insensitive to the presence of superoxide dismutase (SOD) or manitol (Apostol et al.,
1989). These and other observations indicate that O2., and more so H2O2, must be intimately
involved in determining the result of plant-pathogen interactions.
For the verification of this hypothesis, three mechanisms have been proposed:
1. H2O2 is the substrate for many important reactions in the reinforcement of the cell wall,
given that it has been demonstrated that H2O2 leads to oxidative bonds of glycoproteins of the
cell wall, thereby reducing susceptibility of this structure to enzymatic degradation (Bradley et
al., 1992; Brisson et al., 1994; Wojtaszek, 1995). This governs the ability of the pathogen to
penetrate the cell.
2. O2. and H2O2 can act as secondary messengers in the pathway of signals that trigger
genetic activation of PRP (Klessig & Malamy, 1994) and inducing SAR (Chen et al., 1993).
3. Both substances may be actively involved in killing pathogens and/or host cells in HR,
since H2O2 has been demonstrated to be directly toxic for microorganisms (Peng & Kuc, 1992;
Levine et al., 1994; Tenhaken et al., 1995).
In relation to this, the participation of the enzymes involved in the elimination of oxyradicals
and their location, both subcellular and exocellular, play a critical role in determining the con-

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sequences of plant-pathogen interactions. Thus, in the roots of tomatoes infected with a compatible strain of nematode Meloidogyne incognita, SOD activity increases considerably in comparison with that of resistant varieties that manifest the HR and that do not induce SOD (Zacheo
& Bleve Zacheo, 1988; Zacheo et al., 1983). This fact appears to indicate that in an incompatible reaction the low SOD activity enables the O2. and related oxygen species to destroy plant
cells, leading to an HR, whereas in a compatible reaction the high SOD activity assures the
detoxification of oxyradicals and increases less-localized cell death (Bowler et al., 1992).
Also, if O2. and H2O2 act as secondary messengers, the changes in SOD activity may alter
their ability to transduce the signal that controls quantities within the cell, both in compatible
reactions and incompatible reactions. Thus it has been reported that, in tobacco leaves infiltrated with benzoic acid activated with H2O2 and the enzyme 2-hydroxylase (the enzyme that
forms SA from benzoic acid), the result is an accumulation of SA (Leon et al., 1995). In turn,
SA reportedly inhibits CAT and ascorbate peroxidase (APX) and also regulates AOS levels
(Durner & Klessig, 1995).
Finally, it is known that structural proteins that are insolubilized after reaching the cell wall
result from a reaction catalyzed by a peroxidase from the cell wall (Smith-Becker et al., 1998).
These proteins are insolubilized through covalent bonds, and this action has been shown to be
led by H2O2 (Wojtaszek et al., 1995). In plants this rapid protein immobilization appears to
reinforce the cell wall, thereby slowing down the entry of the pathogen (Brisson et al., 1994).
Also, this immobilization may act as an anchor in the immobilization of phenolic compounds
and the consequent formation of papillae (Wojtaszek et al., 1995).
The development of oxidative metabolism in plants begins as a response to a large number
of factors, both biotic and abiotic (Mehdy et al., 1996). Among the diverse environmental
factors that can affect the activities of these enzymes are luminance intensity (Mishra et al.,
1995), exposure to herbicides (Aono et al., 1995), air pollutants (e.g., O3, SO2; Rainieri et al.,
1996), cold (Zhang & Kirkham, 1995), drought (Olsson et al., 1996), waterlogging (Monk et
al., 1987), salt toxicity (Hernndez et al., 1995), and heavy-metal toxicity (Weckx & Clijters,
1996). Also, these enzymatic activities can be altered by certain nutritional imbalances in elements such as zinc (Zn), manganese (Mn), Fe, Cu, Mg, K, and B (Cakmak & Marschner, 1993;
Iturbe-Omaetxe et al., 1995; Wenzel & Melhorn, 1995).
Due to the essential role of oxidative metabolism in the development of plant resistance to
pathogen attack, and given the importance of fungicides in combatting plant disease, Garca et
al. (unpubl. data) determined the effect of the application of different dosages of carbendazim
on the H2O2 levels as well as on the activity of related enzymes, such as SOD, GPX, APX, and
CAT, in tobacco plants. Their results indicated that again the application of the carbendazim
dosage of 50% lower than recommended (carb 1; 1.3mM) may be more effective, since the
foliar accumulation of H2O2, together with the highest biomass production registered for carb
1, may strengthen the plants resistance to pathogen infection. On the other hand, the authors
found that excessive application of carbendazim (carb 3; 5.2 mM) appreciably limited the
capacity of these plants to respond to pathogen attack, since the biomass as well as H2O2 accumulation declined significantly in this treatment. These findings show that in tobacco plants
the defense mechanisms innate to plants contribute to the effectiveness of carbendazim as long
as this fungicide is applied only at the appropriate concentration.

V. Conclusions
In conclusion, the idea that fungicides are well tolerated and that they are compatible for
plants leads us to conclude that there are no direct effects or inhibition from the treatments with

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these compounds. Thus a detailed study of the effects of fungicides, together with research on
the synthesis of appropriate chemical products, should help in the selection of more effective
protective chemicals that are less toxic and less ecologically harmful.
In summary, we believe that work on the effects of fungicides on plant physiology, particularly those of carbendazim, constitutes a valuable line of research having as its main goal to
improve plants defense capacity against pathogen attack. In this regard, we propose that future
work focus on:
1. Verifying whether there is an effect, either direct or indirect, of other benzimidazoles or
systemic fungicides apart from carbendazim, on the key stages of phenolic and oxidative metabolism. In this way, the substances chosen could be tested for their impact on the defense
systems against pathogen attack in plants.
2. Selecting optimal treatments that activate these metabolic processes more efficiently, in
order to provide preventive measures against infection and to examine the effectiveness of
reducing the fungicide application rate normally used in agricultural.
3. Evaluating the possible toxic effects on the physiology of the plant caused by fungicides
when applied at high concentrations and assessing the economic losses as well as dangers to
human health and the environment.

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