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Genesis of azole antifungal resistance from agriculture to clinical settings Maria Manuel Azevedo, Isabel Faria-Ramos, Luísa Cruz, Cidália Pina-Vaz, and Acácio Gonçalves Rodrigues J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b02728 • Publication Date (Web): 19 Aug 2015 Downloaded from http://pubs.acs.org on August 25, 2015

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Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Journal of Agricultural and Food Chemistry

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Title. Genesis of azole antifungal resistance from agriculture to clinical settings

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Maria-Manuel Azevedo1,2,3, Isabel Faria-Ramos1,2, Luísa Costa Cruz1, Cidália Pina-

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Vaz1,2,4, Acácio Gonçalves Rodrigues1,2

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Portugal.

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Medicine, University of Porto, Porto, Portugal

Department of Microbiology, Faculty of Medicine, University of Porto, Porto,

Center for Research in Health Technologies and Information Systems, Faculty of

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School D. Maria II, Rua da Alegria, 4760-067, Vila Nova de Famalicão, Portugal.

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Department of Clinical Pathology, Centro Hospitalar de São João, Porto, Portugal.

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Corresponding author

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Maria Manuel Azevedo

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Department of Microbiology, Faculty of Medicine, University of Porto, Porto, Portugal.

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E-mail: [email protected]

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Telefone:+351936320962

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ABSTRACT

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Azole fungal resistance, is becoming a major public health problem in medicine in

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recent years. However, it was known in agriculture since several decades; the extensive

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use of these compounds results in contamination of air, plants and soil.

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The increasing frequency of life-threatening fungal infections and the increase of

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prophylactical use of azoles in high-risk patients, taken together with the evolutionary

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biology evidence that drug selection pressure is an important factor for the emergence

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and spread of drug resistance can result in a dramatic scenario.

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This study reviews the azole use in agricultural and in medical context and discusses

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the hypothetical link between its extensive use and the emergence of azole resistance

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among human fungal pathogens.

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KEYWORDS: antifungals, azoles, fungal resistance, Candida, Aspergillus fumigatus,

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Portuguese agriculture

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INTRODUCTION

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The use of plant protection products (PPP) may have considerable negative effects on

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the environment 1. The use of azole fungicides in agriculture started around mid-1960s;

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since then, such compounds have been used extensively 2. In the European Union half

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of the total land of cereals and grapevine is treated with azole fungicides. Notably, much

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smaller quantities of azoles are used in the United States for plant protection3.

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The azoles used in agriculture are generally sprayed every year over the cultured area in

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order to control mildews, rust and other diseases in cereals, ornamental plants,

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vegetables, fruits and vineyards. According to Matthews, the widespread use of these

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compounds results in contamination of air, plants and soil with a lot of small particles of

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fungicide4.

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The hypothetical risk that the massive use of azoles in agriculture could induce

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antifungal resistance among human pathogens started to be discussed several years ago.

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Agricultural azoles could stress fungal species either members of the endogenous

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saprophytic flora or exogenous to the human body. The imbalance of fungal ecology

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could thus upset the population of medically important fungi. Human pathogenic fungi

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may therefore persist and thrive5, and in this situation, health risks for human beings

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increase.

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In order to address this problem, in particular regarding the Portuguese situation

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relatively to azole use in agriculture, this review was organized according the following

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topics: 1. Use of azole fungicides in agriculture; 2. Use of agricultural azole fungicides

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in Portugal; 3. Mode of action of drugs; 4. Hypothetical link between azole use in

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agriculture and human health; 5. Portuguese National Strategie plan for a sustainable

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use of plant protection products.

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Use of azole fungicides in agriculture

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The growing request for food in quantity/quality has placed a drastic pressure on the

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productive sector, often at the expense of the natural elements, with progressive

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reduction or elimination of pristine landscape. The use of PPP in this context may have

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strong negative effects on the environment, especially with regard to the possible

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contamination of surface water and groundwater. Azole compounds are PPP which are

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also used for preservation of materials, such as paints, coatings, wall paper paste, and

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are routinely applied to mattresses in order to prevent fungal growth. The most

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commonly compounds used in the European Union are imidazoles6 (Figure 1A)7 and

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triazoles (Figure 1B)8. They are widely used in preharvest phase, in grain-growing and

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grass-growing environments, as well as at postharvest phase to prevent fungal spoilage9

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by yeasts like Candida spp., Cryptococcus spp, Rhodotorula spp., Trichosporon

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penicillatum, Saccharomyces cerevisiae or filamentous moulds like Alternaria spp.,

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Aspergillus spp., Fusarium spp., Geotrichum spp10,11. In addition, they are also applied

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to treat plant infection when it becomes apparent.

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According to the manufacturer’s instructions, doses of 100g/ha should be used,

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corresponding approximately to 10 mg of azoles applied to 1 m2 of plant surface5. Data

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from the literature reveals that annually nearly 50% of the total acreage under cereal and

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grapevine production in Europe is treated with azole fungicides12. Comparatively, in the

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United States, less than 5% of the total crop area is treated with these compounds. In the

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European Union, the situation of the Netherlands stands out in particular, since the

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utilization of azole fungicides has almost doubled since the mid-1990s13. Several

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reasons are usually forwarded to justify the use of azoles in agriculture, in particular, the

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fact that these compounds are not expensive and that they exhibit broad spectrum

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activity. However, since azoles are very stable molecules, they can persist active in the

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soil and water during several months14 as well as in several fruits and vegetables15,16,17.

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Although the azole residues detected in many samples do not reach health hazards toxic

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levels, the amount could vary considerably. Data from the literature shows that

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considerable quantities of azole residues could persist in some food products during

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long time5.

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Farmers have been strongly encouraged to follow guidelines aiming to reduce the

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probability of developing resistance to agricultural antifungals and/or to minimise its

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extent and/or to increase the chance that any developed resistance would still be

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reversible. Such practices include the rotating use of antifungal products with different

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modes of action, as well as the preferential practice of a limited number of interventions

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using higher dosages as opposed to more frequent applications with lower dosages. This

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does not, however, imply that the use of antifungal substances in agriculture should not

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be closely monitored, considering the risk of accumulation of azoles in the soils (the

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half-life time of azoles is usually longer than 1 year), and because the newer compounds

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may be also at the origin of resistance development.

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Use of agricultural azole fungicides in Portugal

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In the particular case of Portugal, there is a real need to promote agriculture and invest

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in training farmers. According to the results of the Agricultural Census report18, the

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social characterization of the Portuguese family farm population consists of 793 000

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individuals representing, 7% of the resident population. The rural population aged

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considerably between 1999 and 2009; the average age increased from 46 to 52 years.

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The educational level is rather low; 40 % of subjects only attended the 1st cycle and

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22% have no schooling. However, despite these social indicators, significant

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improvements were registered during the ten year period under study; illiteracy rate

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decreased by 7% and the frequency of senior secondary cycle and higher education

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increased by 3%. At the present, the typical profile of the Portuguese farmer nowadays

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is a 63 year old male, having just completed the 1st cycle of basic education, only

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having practical agricultural training and working in agricultural activities about 22

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hours per week.

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By December 31, 2012, 907 PPP, based on 248 active substances were available in

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Portugal19. The largest volume of these substances available at the national market

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belong to the fungicides group, followed by herbicides and insecticides, representing the

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remaining products about 15% of the total PPP marketed. According to the Agricultural

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Statistics report the ratio between sales of PPP/agricultural area used was in 2008, 2009,

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2010 of 4.6, 3.8 and 3.8, respectively. In case the total sales volume is subtracted with

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the value of sulfur and derivates, this ratio corresponds to values of 1.9, 2.0, and 1.9,

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respectively19.

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Sales figures of PPP at national level constitute an indicator that can provide an

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estimation about the use of such products in Portugal. The amount of PPP sold in

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Portugal during 2012 corresponded to a total of 12 462237 kg, expressed as active

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substances. The fungicides contributed with about 68.3% of the total of active

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substances sold, whereas sulphur represented 49.8 % of the total sales and 71.4% of all

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the fungicides sold. Figure 2 depicts an overview of fungicide sales by chemical group

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in Portugal between 2002 and 2012. Overall, a decrease in fungicide sales was

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registered from 2008 until 201220 . In respect to azoles, a consecutive decrease in sales

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was registered between 2002 and 2005; however, after 2006 the values started to

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increase again, reaching a peak in 2012 (Figure 3)20.

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Mode of action of azole drugs

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Concerning the mode of action, all the azole drugs interact and target the same active

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site in fungal enzyme; thus agricultural azole fungicides share the mode of action with

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medical azoles21. This results in a fungistatic rather than fungicidal effect; in this sense

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the terminology often used in agriculture “fungicides” is ambiguous.

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This class of compounds are sterol demethylation-inhibitors (SBIs) and impair the

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synthesis of ergosterol5,21. Azole drugs interfere with the activity of fungal lanosterol 14

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alpha-demethylase, an enzyme responsible for the transformation of lanosterol in

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ergosterol. Ergosterol is an important compound of the fungal cytoplasmatic membrane;

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the impairment of its metabolism induces cell membrane disorganization and decrease

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of fungal growth. Moreover, fungal growth arrest can also result from the accumulation

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of toxic ergosterol precursors22.

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The available SBIs-fungicides can be classified into four classes according to their

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respective target sites, which are membrane bound enzymes, namely: thiocarbamates or

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allylamines (squalene epoxidase), inhibithors of C14-demethylation or DMIs, Amines

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(∆14 reduction or ∆8-> ∆7 – isomerization) and hydroxyanilides (C4-demethylation).

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However, several resistance mechanisms to SBIs have been described. The two major

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involve either alterations of the target enzyme, which result in a reduced affinity to

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fungicides or a decrease of the intracellular drug accumulation, that is linked to an over

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expression of membrane transporter proteins, usually namely effux-pumps.

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Hypothetical link between azole use in agriculture and human health

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Several reports claim that antibiotic resistance can be a natural occurrence in the soil. In

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fact, microorganisms inhabiting soil have to strive for energy sources; it is known that,

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some bacteria and fungi develop the ability to produce antibiotic compounds in order to

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destroy their competitors. Such microorganisms are thus naturally immune to the effects

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of their own excretions23.

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The development of resistance to azoles among plant fungal pathogens is complex and

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generally progressing in small steps. First descriptions date from long time ago and

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claim that some plant pathogens acquired azole resistance following agricultural

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exposure24. Data from the literature describes resistance or tolerance to triazole

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fungicides for pathogens of crops such as wheat, barley and strawberry25. While in

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Portugal resistance to triazoles in vineyard powdery mildew has been detected in the

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past26, still recent studies describe this situation27.

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Over the last two decades, clinically important fungal infections have become more

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prevalent because medical progress allows the survival of an increasing number of

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immunocompromised patients. However, resistance to antifungal drugs also became a

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critical medical problem.

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It has been proposed, that there may be a direct relation between the development of

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resistance to azole fungicides used in agricultural practice and the development of

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resistance to azole antifungals observed in clinical settings28. Exposure of a

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microorganism to any antibiotic involves a risk of emergence of resistance through the

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process of selection. The knowledge about the rate and extent of the emergence of

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resistant organisms29 and whether this resistance is reversible or not, is a very relevant

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issue to clinical medicine. Resistance depends upon a variety of factors and conditions,

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including: the mechanisms of action of the compound; the target site(s) in the organism

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and their number; the risk of resistance transference between individual organisms and

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species; and the mechanism of such resistance.

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Resistance in fungi and the mechanisms involved in its development and transmission

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differ in some extent from those seen in bacteria. There is no evidence that the genes

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that confer resistance to fungi can be transferred horizontally, a key difference with

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bacteria. There are two forms of resistance, primary (or intrinsic) and secondary. In

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primary resistance, the fungal organism cannot be inhibited even by high concentrations

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of an antifungal drug. For example, Aspergillus fumigatus is intrinsically resistant to

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fluconazole and does not respond at all to this drug. Similarly, Candida krusei and C.

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glabrata isolates are often intrinsically resistant to fluconazole. Conversely, fluconazole

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resistance in Candida albicans can emerge de novo during prolonged treatment; which

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is an example of secondary resistance30.

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There are at least three different mechanisms by which antifungal drug resistance might

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arise. They can involve increased efflux of drug, altered target demethylase sites and the

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availability of alternative pathways for the synthesis of cell membrane sterols. Candida

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krusei shows intrinsic resistance to fluconazole largely due to the last mentioned

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mechanism.

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There is also evidence that in clinical settings the adoption of some therapeutic

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strategies to treat patients over long periods or to prevent infection through antifungal

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prophylaxis can result in selection of resistant organisms, either of the same or of

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different species. Several studies have shown that a strong and persistent antimicrobial

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pressure upon a microbial population will undoubtedly lead to the selection of resistant

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clones, particularly whenever the antimicrobial agent induces a static effect instead of a

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biocidal effect23. This situation can happen in fungi submitted to azole pressure. Since

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azoles represent first line clinical antifungals, the topic of azole resistance is of great

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medical relevance. Azole resistance can thus hypothetically result from exposure to

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azole compounds, which can happen in vivo (azole treated patients) or due to the

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presence of these compounds in the environment, namely agriculture use31. The massive

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use of azoles in agriculture could stress susceptible species of the fungal flora, both in

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nature but also at human endogenous niches. This disturbance in the fungal flora

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ecology could result in changes among the population of medically important fungi;

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human fungi pathogenic may thus persist and thrive 5. In this situation, health risks

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might increase seriously. In addition, new resistance mutations could also develop in

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the environment. Studies addressing the epidemiology of invasive fungal infections are

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of high relevance in order to determine the hipothetical impact of anthropogenic

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activities upon the susceptibility profile of yeast and filamentous fungi and also to

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report any emergent species. In Portugal, two studies conducted at the same institution,

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within a ten year interval, revealed several relevant differences not only in susceptibility

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patterns but, most importantly, regarding species distribution29,32.

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Invasive aspergillosis is a very dangerous opportunistic fungal infection. The conidia of

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Aspergillus spp. usually become airborne from soil or decaying organic matter and

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patients become infected usually by inhalation from such a reservoir13. In 2009,

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Snelders and co-workers provided for the first time evidence that patients with invasive

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aspergillosis due to azole resistant A. fumigatus might have acquired the organism from

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the environment31. According to this study, resistant strains of A. fumigatus to medical

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azoles were found in the soil and compost from areas nearby to the admission hospital31.

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The dominance of a single resistance mechanism and the genetic homology between the

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clinical and environmental isolates suggested that the acquisition of azole-resistant

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isolates from the environment was the most important infection route.

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Azole resistance among A. fumigatus isolates was not only found in Dutch hospitals33;

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resistant isolates were also detected in several European countries such as in Spain34,

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Belgium35, Denmark36 , Sweden37 and France38.

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Human and animal fungal pathogens such as Coccidioides, Histoplasma, Aspergillus

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and Cryptococcus do also thrive in the environment, including plants and food products.

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It should also be stressed that only a few yeast species are endogenous to the flora of

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healthy human beings; in most occasions, the fungal pathogens are taken up from the

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environment. Thus, environmental resistant isolates could have developed antifungal

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resistance following a prolonged environmental selective azole pressure. Therefore, the

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probability that an individual might be exposed to an environmental resistant fungal

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organisms is real and high. Nevertheless, the stress of residual azoles that might be

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present in vegetables and other food products upon the endogenous native fungal flora

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of humans should not be neglected and may also contribute to the emergence of

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antifungal resistance at endogenous niches.

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In the case of A. fumigatus, it was concluded that two distinct models of resistance

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might have developed. One of them occurs following long time-exposure to azole

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treatment, as might occur in clinical settings, supported by several possible genetic

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mutations, the other model is facilitated by a single mechanism, TR/L98H mutation,

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that occurs in environmental isolates under the selective pressure of agricultural azole

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fungicides. This kind of mutation started spreading worldwide and has nowadays also

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been described outside Europe39. A very recent study conducted in France describes a

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clinical case of a French farmer who developed invasive aspergillosis caused by an

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azole-resistant A. fumigatus with the TR34/L98H mutation following hematopoietic

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stem cell transplantation40. This farmer had worked in fungicide-sprayed fields from

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where A. fumigatus TR34/L98H isolates were also recovered. This study supports the

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assumption that humans might indeed be infected with environmental azole resistant

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isolates.

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Measures in order to control this problem should be adopted, in particular in the

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Netherlands, where the TR/L98H mutation became endemic5. Interestingly, in Portugal,

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back in 2007 Araújo et al.41 compared the antifungal susceptibility profile of different

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Aspergillus species recovered from environmental and clinical sources and found high

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MIC levels of clinical antifungals among environmental strains.

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Verweij and co-workers reported cross-resistance between azole drugs, which

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constitutes a very relevant clinical issue, with relatively few therapeutic options

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available in the case of invasive Aspergillus infections42 . More recently, several studies

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demonstrated the development cross resistance between agricultural and medical

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azoles13,21. Faria-Ramos and co-workers,43 clearly demonstrated that exposure of

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clinically relevant moulds and yeast organisms to azoles used in agriculture does result

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in the emergence of cross-resistance to clinical azoles, which may not be reversible.

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Additional research efforts aiming to reduce the emergence of fungicide resistance are

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urgently needed, both in agriculture and in medical settings. Since the mechanisms

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providing the basis for fungicide resistance are likely to be conserved between plant and

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human pathogens, plant pathologists and medical microbiologists should develop

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effective strategies counteracting the development of antifungal resistance, in a

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concerted action.

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Portuguese National Strategie Plan for a sustainable use of plant protection

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products

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The Directive No. 2009/128/EC is an innovation in the context of the Community

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legislation about PPP; for the first time legislation was provided about the use of PPP,

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in order to protect human health and the environment from possible risks inherent to its

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use.

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In order to operationalize the implementation of this Directive at national level, the law

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nº. 26/2013 was published; it regulates the distribution, sales and application of PPP by

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professional users as well as adjuvants for PPP; it also sets the monitoring procedures

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for the use of PPP. Together with the Law nº. 86/2010 (which establishes a mandatory

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inspection scheme of PPP and its application using equipment authorized for

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professional use) constitutes the transposition into national legal order of that

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Community law.

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The article 51º of this law envisaged the creation of National Action Plans. These plans

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aimed at reducing the risks and effects of the use of PPP on human health and upon the

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environment, and to promote the development of integrated protection technical

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alternatives to reduce dependence of the use of conventional PPP.

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The Portuguese National Plan has been prepared with the collaboration of various

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agents, either from public or private sector, without which would not have been possible

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to achieve the intended goals. It must also be pointed out that the implementation of this

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plan will only be effective with the committed collaboration of all the agents involved

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in manufacture, storage, sale and use of PPP, as well as those responsible for managing

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the effects and risks associated with the use of PPP. The correct application of PPP can

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be encouraged by the training of professionals, appropriate counselling at the point of

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sale, and monitoring/supervision of compliance by professional users. In the act of sale,

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adequate information about the use of PPP and the risks and safety instructions

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regarding human health, should be provided to buyers, in order to enable the adequate

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management of the risks inherent to the products concerned.

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The protection of professionals configures primarily a matter of safety and health

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regarding to the professional handling field use and application of pesticides. Risks may

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include not only acute intoxication, but also chronic and sub-chronic risks, resulting

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from prolonged exposure. The lines of action chosen for this intervention area are: i)

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Protection of professional users (commercial and storage settings); ii) Reduction of

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exposure; iii) Limitation of the use of certain PPP44.

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The main measure of protection regarding non-professional users is restricting its access

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to a limited category of PPP. In any case, buyers should be provided awareness of the

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risks associated with PPP use and about promotion of good general practices regarding

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its use.

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In order to assess the progress made in reducing the risks and adverse effects of the use

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of PPP, both to human health and the environment, monitoring plans should be in place

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so that, based on a sample of professional users and predefined criteria, it will be

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possible to assess the progresses resulting from the implementation of the general

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principles of integrated protection.

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In addition, is should be emphasized the relevance of the contribution of scientific

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research and field application of the most recently available scientific and technological

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information in improving resources and instruments available to the professional users,

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including complementary or alternative means of use of PPP.

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In conclusion, fungicide-based plant protection is at present indispensable for efficient

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and large-scale crop production. Notably, modern fungicides should exhibit negligible

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acute toxicity, and the regulations for fungicide application should ensure the

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availability of safe food. As long time periods and enormous financial investments are

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required to develop new fungicides, serious efforts must be taken in order to prevent

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emergence of fungicide resistance. This is particularly important since fungicides with

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novel modes of action are rarely found, and resistance to single-target fungicides may

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occur within the first few years of its field use.

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The key to prevent or at least delay the onset of development of fungicide resistance in

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agriculture is the preparation of efficient fungicide mixtures as well as the rotation

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between fungicides with different modes of action during a crop campaign. The

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recommendations are to maintain highly efficient fungicides in the market, precluding

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the use of monofungicides, in order to keep its use above a threshold allowing

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quantitative resistance to develop. The strategy adopted during the last decades was to

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apply single-target fungicides, resulted in a strong selective pressure for mutants with a

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modified fungicide-binding site. As fungicides with more than one target are not easy to

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be overcome by mutations, the focus in fungicide screening should also be directed

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towards identifying multi-site inhibitors. After having such fungicides available, the risk

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of rapid occurrence of qualitative resistance might be strongly and effectively reduced.

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Nevertheless, particular attention should be devoted to the assessment of development

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of cross-resistance to clinical antifungals; strict monitoring of the emergence of fungal

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strains with resistance mechanisms, as well as variations in species distribution

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regarding both plant and human pathogens should be implemented.

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CONFLITCTS OF INTEREST

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The authors declare no conflict of interest.

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ACKNOWLEDGMENTS

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I. Faria-Ramos is supported by an FCT PhD grant (SFRH/BD/91155/2012).

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_pt.pdf).

-Volume

518 519 520 521 522

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LEGENDS

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Figure 1. Chemical struture of imidazole (A), 1,2,4-triazole (B), fluconazole (C) and

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tebuconazole (D).

526 527

Figure 2. Sales of fungicides in Portugal between 2002 and 2012 (Source: DGADR –

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Direção Geral de Agricultura e Desenvolvimento Rural “Vendas de Produtos

529

Fitofarmacêuticos em Portugal 2002-2012” http://www.dgadr.mamaot.pt/).

530 531

Figure 3. Sales of agricultural azole in Portugal between 2002 and 2012 (Source:

532

DGADR – Direção Geral de Agricultura e Desenvolvimento Rural “Vendas de Produtos

533

Fitofarmacêuticos em Portugal 2002-2012” http://www.dgadr.mamaot.pt/).

534 535 536 537 538 539 540 541 542 543 544 545 546 547

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Figure 1

A

B

C

D

551 552 553

Figure 2

Sales of azoles for plant protection in Portugal (kg of active substance)

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60000 50000 40000 30000 20000 10000 0 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 Years

555 556 557 558 559 560 561 562 563 564

Figure 3

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Sales of fungicides for plant protection in Portugal (kg of active substance)

24

14000000 12000000 10000000 8000000 6000000 4000000 2000000 0 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 Years

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208x172mm (150 x 150 DPI)

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Figure 1. Chemical struture of imidazole (A), 1,2,4-triazole (B), fluconazole (C) and tebuconazole (D). 254x190mm (96 x 96 DPI)

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