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Article

Efflux Pumps and Multidrug-Resistance in Pyricularia oryzae Triticum Lineage

by
Samara Nunes Campos Vicentini
1,
Silvino Intra Moreira
1,
Abimael Gomes da Silva
1,
Tamiris Yoshie Kiyama de Oliveira
1,
Tatiane Carla Silva
1,
Fabio Gomes Assis Junior
1,
Loane Dantas Krug
1,
Adriano Augusto de Paiva Custódio
2,
Rui Pereira Leite Júnior
2,
Paulo Eduardo Teodoro
3,
Bart Fraaije
4 and
Paulo Cezar Ceresini
1,*
1
Department of Crop Protection, Agricultural Engineering and Soil (Departamento de Fitossanidade Engenharia Rural e Solos), Sao Paulo State University, UNESP, Ilha Solteira 15385-000, SP, Brazil
2
Paraná Rural Development Institute, IDR—Paraná/IAPAR, Londrina 86047-902, PR, Brazil
3
Campus CPSC, Federal University of Mato Grosso do Sul, UFMS, Chapadão do Sul 79560-000, MS, Brazil
4
National Institute of Agricultural Botany, NIAB, Cambridge CB3 0LE, UK
*
Author to whom correspondence should be addressed.
Agronomy 2022, 12(9), 2068; https://doi.org/10.3390/agronomy12092068
Submission received: 29 June 2022 / Revised: 4 August 2022 / Accepted: 5 August 2022 / Published: 30 August 2022
(This article belongs to the Special Issue Evolution-Smart Integrated Disease Management Strategies against Fungicide Resistance in the Agroecosystem)
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Abstract

:
Widespread resistance to QoIs, DMI and SDHIs fungicides has been reported for Brazilian populations of the wheat blast pathogen Pyricularia oryzae Triticum lineage (PoTl). A pre-existing resistance mechanism not associated with target site mutations has been indicated for resistance to DMIs and SDHIs, with strong indication that PoTl has multidrugresistance (MDR). Therefore, the main objective of this study was to test the hypothesis that resistance to DMI and SDHI fungicides detected in PoTl was due to efflux pump mediated MDR mechanism(s) by characterizing the sensitivity to antifungal efflux pump substrates. Four antifungal substrates were tested: tolnaftate (TOL), cycloheximide (CHX), rhodamine 6G (RH6G) and triphenyltin chloride (TPCL). TPCL and RH6G were considered the most relevant indicators for enhanced MDR activity. Among the 16 PoTl isolates tested, 9 were insensitive to TPCL, 1 to TOL, 16 to RH6G and 1 to CHX. The PoTl isolates were grouped into four distinct multidrug resistance phenotypes (MDRPs) based on resistance to combinations of fungicides and antifungal efflux pump substrates. Insensitivity to TPCL, RH6G and or TOL correlated well with DMI insensitivity, but MDR was not associated with SDHI resistance. The identification of multiple MDRP phenotypes associated with DMI resistance in our study warrants further research aimed at revealing the exact mechanisms of multidrug resistance in the wheat blast pathogen, including efflux pumps overexpression via transcriptomic analyses of differentially expressed genes; identification and discovery of mutations associated with changes in promoter regions or transcription factors of efflux transporters associated with multidrug resistance.
Keywords:
wheat blast; MDR; QoI; DMI; SDHI

1. Introduction

Wheat blast is caused by the ascomycete fungus Pyricularia oryzae Triticum lineage (PoTl) [1,2]. This is a very aggressive disease, and its management is hampered by the absence of durable host plant resistance sources due to the extremely variable pathogen populations, both genetically and phenotypically [3,4]. In addition, the frequent and extensive use of site-specific fungicides without adopting anti-resistance strategies, including other integrated disease management strategies, has resulted in a general loss of fungicide efficacy for management of the disease [2,5].
Resistance to the site-specific fungicides QoI (quinone-outside inhibitors) and DMI (sterol 14α-demethylation inhibitors) has become pervasive and persistent in PoTl populations from all wheat-cropping regions throughout Brazil over a time span of seven years, from 2012 to 2019 [6,7,8,9,10]. Resistance to QoI was mainly associated with cytB target site alteration G143A (substitution of glycine (G) by alanine (A) at codon 143), which has been reported for many plant pathogens [6,11]. Due to the absence of CYP51 mutations and promoter inserts linked with azole resistance, the mechanism(s) conferring azole resistance remains unclear for PoTl populations in Brazil.
Moreover, resistance to the new generation carboxamide fluxapyroxad (a succinate dehydrogenase (Sdh) inhibitor (SDHI) fungicide) was detected in PoTl populations sampled in 2012 and 2019 [10]. Since SDHI resistance was detected in Brazil before the introduction of this fungicide MOA in the cereals market and no changes in Sdh subunits B, C and D, forming the SDHI binding site, were detected, it was suggested that resistance is mediated by a pre-existing non-target site resistance mechanism.
Reduced sensitivity phenotype to multiple chemically unrelated compounds is defined as multidrug resistance (MDR), which is often caused by overexpression of efflux transporters with broad substrate specificity, as has been reported for a range of plant pathogens [12,13,14,15]. In fungi, the major drug efflux pumps are members of the ATP-binding cassette (ABC) and major facilitator superfamily (MFS) transporter proteins [16]. Together, these two superfamilies of integral membrane transporters represent roughly half of all the genes coding for transporters in fungal genomes [17]. As an example, Zymoseptoria tritici genome analysis indicated the presence of 47 ABC transporters and 288 MFS transporters, of which at least 87 of the latter are putative multidrug transporters [18].
ABC transporters consists of transmembrane domains (TMD) and structurally conserved nucleotide-binding domains (NBDs) and function as molecular machines by coupling ATP binding, hydrolysis, and phosphate release to the translocation of diverse substrates across membranes [19]. In comparison, MFS transporters use proton motive forces to drive substances across membranes. These proteins play fundamental roles, such as the transport of small molecules, metabolites, and ions, as well as fungicides, toxins, and heavy metals removal as protection against biotic and abiotic stresses [19,20,21]. ABC or MFS transporters are located in different types of membranes, from the external plasma membrane to intracellular compartments membranes such as vacuole, endoplasmic reticulum, peroxisomes and mitochondria [16,19,22,23].
Overexpression of efflux pumps can be caused by modifications in their promoter regions or due to gain-of-function mutations in transcription factors that control expression. Omrane et al. [24] identified for the Septoria leaf blotch pathogen Z. tritici a 519-bp long terminal repeat (LTR) insert (relic of transposon activity) in the MFS1 promoter co-segregating with the MDR phenotype using a bulk progeny sequencing approach. Through gene replacement, they showed that the promoter insert was responsible for MFS1 overexpression and the MDR phenotype. In addition, different types of shorter promoter inserts were found in more recent MDR strains. In MDR strains of human pathogen Aspergillus fumigatus, the C2H2 zinc finger transcription factor SltA was pointed as relevant for azole resistance by coregulating the expression of the fungicide target gene CYP51A and the efflux pump Mdr1 [25]. In MDR isolates of the rice blast pathogen PoOl, single point mutations in the MoIRR gene encoding a Zn2Cys6 zinc finger transcription factor-like protein confers resistance to isoprothiolane and to iprobenfos plus tricyclazole [15]. This Zn2Cys6 domain is also present in the Mrr1 transcription factor found in the opportunistic human fungal pathogen Candida albicans and in grape’s gray mold pathogen Botrytis cinerea [13,26]. Specifically, mutations in the Mrr1 regulates gene expression of CDR1 (ABC) transporter in C. albicans and the BcAtrK (ABC) transporter in B. cinerea with MDR phenotype MDR1. Another mechanism reported for MDR2 in B. cinerea is the BcmfsM2 (MFS) transporter gene promoter region rearrangement [13,27]. MDR3 in B. cinerea, generated through sexual crossing, combines both the MDR1 and MDR2 mechanisms, resulting in increased substrate range, and resistance to fludioxonil, cyprodinil and tolnaftate.
In general, multidrug transporters have a wide range of substrates with differences in specificity roles according to transporter types, their abundance and recognition of a domain’s structure. For example, the MgMFS1 transporter system in Z. tritici has redundancy roles in the transport of rhodamine 6G, once its gene deletion is compensated by expression changes in other transporters with an affinity for this drug [28]. However, the knockout of MgMFS1 caused an increase in sensitivity to QoI fungicides, with no redundancy in this case. In fact, there is no obvious correlation between homology in the transporter amino acid sequence and specificity to substrates. For instance, the PoOl ABC2 transporter has high amino acid homology with BMR1 (BcatrK) in B. cinerea, although they have different ranges of substrates [29]. The PoOl ABC2 transports the DMI tebuconazole, cycloheximide, bitertanol, myclobutanil and camptothecin, while BMR1 extrudes ibrobenfos and polyoxin.
Chemically unrelated antifungal compounds have been used as efflux pump substrates in order to test hypotheses about the role of MDR mechanisms and to distinguish different MDR phenotypes caused by enhanced activity of MFS and/or ABC transporters. Triphenyltin chloride, tolnaftate, rhodamine 6G and cycloheximide are amongst the most common efflux pump substrates used as indicators of MDR phenotypes [14,30,31,32]. In our study, we tested the hypothesis that resistance to DMI and SDHI fungicides detected in PoTl was due to efflux pump mediated MDR mechanism(s) by characterizing the sensitivity to these four antifungal efflux pump substrates. Based on the sensitivity levels to the efflux pump substrates, we can also identify if there are different MDR phenotypes amongst the PoTl strains previously sampled in 2012 and 2018.

2. Materials and Methods

2.1. Fungal Strains

To carry out this study, twenty-one isolates of Pyricularia spp. previously classified according to their insensitivity levels to QoI, DMI and SDHI fungicides were randomly selected from our collections spanning 2007, 2017, 2018 and 2019 sampling years [6,9,10]. The strains selected harbor distinct combinations of resistance phenotypes from fully sensitive isolates to fully resistant isolates to DMI and SDHI fungicides.

2.2. Sensitivity to Efflux Pump Substrates

The antifungal efflux pump substrates tested were tolnaftate (TOL), cycloheximide (CHX), rhodamine 6G (RH6G) and triphenyltin chloride (TPCL) (all from Sigma-Aldrich, St. Louis, MO, USA). These drugs were dissolved to 1 mg·mL−1 in acetone followed by dilution in deionized water to obtain a 100 µg·mL−1 stock solution.
Sensitivity tests with efflux pump substrates were conducted with flat-bottomed 96-well microtiter plates (Kasvi, India) using mycelial fragments. Mycelium fragments were obtained from fungal colonies that were grown for 10 days in PDA at 25 °C under 12 h photoperiod using a protocol adapted from Brito et al. [33] and Arango Isaza et al. [34]. Pathogen mycelial samples from 10-day old colony from a single agar plate were transferred to a mortar and macerated using a pestle in 3 mL sterilized water and Tween 20 (one drop L−1). Each 1 mL of the macerate was transferred to tubes containing a volume of 1 mL of sterile 2 mm glass beads and vortexed for two minutes. The mycelial fragments suspension recovered from tubes were mixed and pre-diluted to a final volume of 15 mL, counted under a microscope using a Neubauer chamber and adjusted to 105 fragments mL−1. Each microplate well was filled with 50 μL of inoculum suspension and 100 μL of PD broth (20.7 g·L−1 of potato dextrose (Kasvi, India), 1 L of distilled water)) amended with different concentrations of the efflux pump substrates tested and in the presence of chloramphenicol and streptomycin (50 μL·mL−1 each).
Stock solutions from each of the substrates were added to the wells to obtain the following final concentrations: tolnaftate: 0.0, 0.032, 0.16, 0.8, 4 and 20 µg·mL−1; cycloheximide, rhodamine 6G and triphenyltin chloride: 0.0, 0.016, 0.08, 0.4, 2 and 10 µg·mL−1. For strains extremely sensitive to triphenyltin chloride, the following dilution series was used: 0.0, 0.000128, 0.00064, 0.0032, 0.016 and 0.08 µg·mL−1. The microplates were incubated at 25 °C for 5 days under a 12 h photoperiod. After incubation, mycelial growth (mg) was measured based on absorbance values at 620 nm (mg = t5 − t0; where t0 = reading (absorbance at 620 nm) immediately after plate assembly; t5 = reading after five days incubation) using a microplate reader (MultiskanTM FC Microplate Photometer, Thermo Scientific TM, Waltham, MA, USA).
Sensitivity to efflux-pump substrates was determined as 50% effective concentration to inhibit fungal growth (EC50 in µg·mL−1), estimated using the software ED50 plus v1.0 [35]. Resistance factors (RF) were determined to estimate the relative fold-inhibition of all the strains tested based on their EC50 values and normalized by the lowest EC50 for each substrate. For EC50 means comparison within each substrate (TPCL, TOL, RH6G or CHX), the experimental design consisted of complete randomized blocks, with four replicates per treatment and experiments in duplicate.

2.3. Statistical Analysis

Analysis of variance (ANOVA) by the F test and means comparison by the Scott-Knott test (at 5% probability) were performed using the R environment with the statistical libraries agricolae and laercio [36].
Pearson’s correlation analysis was performed using EC50 data from all fungal strains to explore the relationships among sensitivity to efflux pump substrates and the fungicides tebuconazole (DMI) and fluxapyroxad (SDHI). The DMI and SDHI EC50 data were obtained from previous studies [9,10]. To verify the correlation significance, t tests were applied using the R environment with the statistical library performance analytics [36].

3. Results

3.1. Sensitivity to Efflux Pumps Substrates

The statistical analysis indicated no significant difference between replicates (R) of the experiments, as well no significant interaction between replicates and the strain (S) factor (R*S). This indicated the reproducibility of the observations allowing for the joint analysis of the replicates. The strain effect was highly significant (p < 0.01) for all four efflux pump substrates checked (Table 1).
Nine out of the 16 PoTl strains tested were insensitive to the efflux pumps substrate triphenyltin chloride (TPCL), as shown by a greater than 5-fold elevated EC50 level in comparison with the average EC50 value of 0.0085 µg·mL−1, recorded for the two fungicide sensitive PoOl strains 421 and 656 (Table 2). The strains PoTl 18SPK6 (Figure 1A), and 18SPC10 showed the highest significant EC50 values for TPCL, 0.31 µg·mL−1, resulting in a resistance factor (RF) of ~36-fold in comparison with the average EC50 value of the two fungicides sensitive PoOl strains (Table 2). The RFs for the other seven TPCL insensitive PoTl strains ranged from 7.1 to 31-fold, while the two other PoTl strains were sensitive, showing RFs < 5.
Only one PoTl strain, 12.1.130 (Figure 1D), was insensitive to tolnaftate (TOL), with an EC50 of 4.40 µg·mL−1, showing a RF of 13 in comparison with the average EC50 value of 0.34 µg·mL−1 measured for the two fungicide sensitive PoOl strains 421 and 656 (Figure 1B and Table 2). All other 15 PoTl strains and 2 PoOl strains tested were sensitive, showing RFs between 0.7 and 3.9.
All 16 PoTl strains and 1 PoOl strain (674) showed raised EC50 values to rhodamine 6G (RH6G) (Table 2). The RFs ranged from 13- to 61-fold for the PoTl strains with the highest EC50 value, 2.13 µg·mL−1, measured for strain 12.1.299 (Figure 1B). A lower RF value of 7.1 was measured for the RH6G sensitive PoOl strain 674.
The cycloheximide (CHX) EC50 values for the 20 Po strains tested ranged from 0.09 to 1.24 µg·mL−1 (Table 2). The fungicide sensitive PoTl reference strain 421 was insensitive and showed a RF of 6.7 in comparison with PoTl strain 656. Only one PoTl strain, 18MGF23, was also insensitive with an EC50 value of 1.24 µg·mL−1 and a RF of 10 (Figure 1C).
The strain P. grisea 363 was resistant only to RH6G, while PoOl 421 was resistant only to CHX (Table 2).
PoTl strains were subsequently classified into multidrug-resistance phenotype patterns (MDRP) based on the resistance levels to the four different efflux pump substrates tested (Table 2). Isolates resistant to either one of the target-site fungicides, DMI or SDHI, or both (Table 2), and also to at least one unrelated efflux pump substrate, were considered as expressing MDR (Table 2). The resistance to QoIs was not used as a parameter, because high levels of resistance in these strains was linked to the target-site alteration G143A in cytochrome b gene.
Four distinct MDR phenotypes (MDRP1-4) were observed amongst the 16 PoTl strains (Table 2): (i) strains with high levels of RH6G and DMI resistance, and with varying levels of SDHI insensitivity (MDRP1, six strains); (ii) strains resistant to RH6G, TPCL and DMI with different levels of SDHI insensitivity (MDRP2, eight strains); (iii) strains resistant to TOL, RH6G, DMI and SDHI (MDRP3, strain 12.1.130) and (iv) strains resistant to TPCL, RH6G, CHX, DMI and SDHI (MDRP4, strain 18MGF23).
PoOl strains 656 and 704 were sensitive to all four efflux pump substrates, but only PoOl 704 was also resistant to the SDHI tested. PoOl strain 421 showed insensitivity to CHX but was sensitive to both DMI and SDHI, while PoOl strain 674 was insensitive to both RH6G and SDHI but sensitive to DMI.

3.2. Correlation Analysis

Positive significant correlation (p ≤ 0.01) was observed among the EC50 values of the fungicide tebuconazole (DMI) and of the substrates TPCL (55%) and RH6G (58%), besides lower significance (p ≤ 0.10) between DMI and TOL (42%) (Figure 2). However, the EC50 values for fluxapyroxad (SDHI) did not correlate with any chemical tested (Figure 2). The sensitivity levels between the substrates TPCL and TOL were significantly correlated (p ≤ 0.001) with high positive linear relationship (close to 80%) (Figure 2). The TPCL also correlated with RH6G (p ≤ 0.01), in which the linear relationship was close to 60%. For TOL and TPCL the correlation was lower than 40% (significance at p ≤ 0.1) (Figure 2). The EC50 values for the efflux pump substrate CHX did not correlate with any chemical tested (Figure 2).

4. Discussion

Resistance to site-specific fungicides in fungi can vary for different unrelated chemicals according to the recognition domain’s structures and the expression patterns of different transporter’s genes, and this variation is population- and strain-associated. Therefore, it is plausible that resistance to the site-specific DMI and SDHI fungicides could occur both due to target-site mechanisms in association with the efflux pump’s overexpression in the MDR isolates [16]. A pre-existing mechanism not associated with target site mutations has been associated with the resistance to DMI and SDHI fungicides, with a strong indication that PoTl has evolved efflux-based multidrug-resistance (MDR).
We tested this hypothesis by testing a selection of 20 Po strains (16 PoTl and 4 PoOl) with different resistance levels to DMI and SDHI fungicides for sensitivity to four antifungal efflux pump substrates, cycloheximide (CHX), triphenyltin chloride (TPCL), rhodamine 6G (RH6G) and tolnaftate (TOL).
CHX is an antifungal and antibiotic isolated from Streptomyces griseus and inhibits protein synthesis. CHX exerts its effect by interfering with the movement of tRNA molecules on the mRNA-ribosomal complex, paralyzing the translocation step of protein synthesis [37]. CHX has been frequently used in efflux pumps studies with transporters such as PoOl ABC2 [29], AtrD in B. cinerea [13] and PdPMR1 in P. digitatum [38]. Yeast heterologous gene expression studies with ABC and MFS transporters from both Z. tritici [18] and C. Albicans [39] show CHX can act as a substrate for both efflux pump families. In comparison with the fungicide sensitive strain PoOl 656, only two strains, PoTl 18MGF23 and PoOl 674, showed raised levels of CHX insensitivity, with RFs of 10 and 6.7, respectively. However, PoOl 674 did not have a MDR phenotype, being sensitive to QoI, DMI and SDHI fungicides, and MDR in the remaining PoTl strains (PoTl strain 18MGF23) is therefore also not likely associated with CHX insensitivity.
Triphenyltin chloride (TPCL) and/or rhodamine 6G (RH6G) can be considered as the most useful indicators of efflux pump mediated MDR activity, since seven strains were insensitive to RH6G alone (MDRP1 and MDRP3) and nine out of 16 fungicides-resistant PoTl strains were insensitive to both RH6G and TPCL (MDRP2 and MDRP4), using a RF of 5 as a cut-off, with a high correlation between their EC50s and the EC50s recorded for the DMI tebuconazole. Fentin compounds such as TPCL inhibit mitochondrial ATPase activity, affecting membrane permeability and calcium influx regulation [40]. Saccharomyces cerevisiae and Z. tritici are examples of fungi screened for MDR using this substrate [32,41]. Rhodamine 6G (RH6G), known to be an inhibitor of mitochondrial oxidative phosphorylation [42], has been used as a transporter substrate to test hypotheses on MDR in many other fungal species, in particular ABC or MFS transporter systems such as C. albicans (Cdr1) [43], Cryptococcus neoformans and C. gattii (Afr1, Afr2, Mdr1) [30]. RH6G is a fluorescent dye with chemical properties appropriate for accumulation or export assays based on fluorescence detection of its activity, which has been applied, for instance, to determine the activity of the ABC transporters CgCDR1 and CgCDR2 in C. glabrata [44].
In our study, only one PoTl strain, 12.1.130, was insensitive to tolnaftate (TOL) (Table 2). This strain, representing MDRP3, was also insensitive to RH6G and showed the highest level of DMI resistance (Table 2). Furthermore, a high level of cross-resistance was found between TOL and RH6G (Figure 2). TOL is a synthetic thiocarbamate used as an antifungal agent, acting as an inhibitor of squalene epoxidase and, consequently, inhibiting the sterol biosynthesis [45]. Tolnaftate has been used for MDR phenotyping in Z. tritici (MgMFS1), B. cinerea (AtrB, mfsM2) and C. albicans (Cdr1 and 2) [13,14,32,45].
In summary, four different MDR phenotypes (MDRP1-4) were observed amongst the 16 PoTl strain tested based on insensitivity levels to multiple fungicides and four antifungal efflux pump substrates (TPCL, TOL, RH6G and CHX). Insensitivity to DMI correlated well with insensitivity levels to TPCL and/or RH6G, and the Pearson’s correlation between the DMI’s EC50 with both TPCL and RH6G EC50 values corroborated this observation.
Regarding DMI fungicides, the widespread reduced triazole sensitivity of the wheat blast pathogen in Brazil could have evolved to its current high levels of resistance in response to intensive exposure of PoTl populations to triazoles over 30 years of selection pressure due to repeated spray applications of DMI fungicides in the local wheat agroecosystem [9]. Although the authors detected five different PoTl CYP51A haplotypes (H1 to H5), four of them all carrying non-synonymous substitutions in CYP51A at high frequencies within field populations, none of these substitutions correlated with elevated EC50 values. For a selection of strains, including PoTl 12.1.299, sequencing of regulatory and coding sequences of both CYP51A and CYP51B revealed no alterations and the operation of a non-target site resistance mechanism was suggested [9]. In our present study this strain was categorized as MDRP2, with resistance to the transporter substrates TPCL and RH6G (Table 2).
In the sister species PoOl, the rice blast pathogen, an ABC2 transporter system was identified as a relevant mechanism for azole resistance. Disruption of genes associated with the ABC transport resulted in increased sensitivity to tebuconazole in PoOl, and also to CHX and other efflux pump drugs [29]. Many other examples of ABC transporters linked to azoles resistance have been reported for different fungi species such as A. nidulans (AtrG), A. fumigatus (Mdr1, abcE), B. cinerea (BcatrB), C. albicans (Cdr1p and 2p), C. neoformans (Afr1p) and Penicillium digitatum (PdPMR1). Moreover, MFS efflux pumps have been associated with DMI resistance in A. fumigatus (mfsC), Z. tritici (MgMFS1), B. cinerea (Bcmfs1) and C. albicans (CaMdr1p) [14,25,46,47,48,49].
Regarding the mechanisms of the resistance to the SDHI fungicide fluxapyroxad, none of the SDHI-resistant PoTl strains harbored any amino acid substitutions in SdhB, C or D [10], also pointing to the existence of a non-target site associated resistance mechanism such as enhanced efflux pump activity. For instance, MgMFS1 promoter insertions conferring resistance to many SDHI molecules, including fluxapyroxad, have been reported for Z. tritici [24], and Mrr1 transcription factor changes altering the ABC-AtrB and the MFS-mfsM2 expression levels have been reported to confer resistance to the SDHI fungicide boscalid [13]. However, one DMI-sensitive PoOl strain without a MDR phenotype, isolate 704, was found moderately resistant to fluxapyroxad (Table 2), whereas three PoTl strains, 12.1.005, 12.1.165 and 18PRH9, showing high levels of resistance to the DMI tebuconazole and classified as MDRP1 or MDRP2 (12.1.005, 12.1.165 and 18PRH9), were sensitive to the SDHI fluxapyroxad (Table 2), indicating another resistance mechanism might be responsible. Interestingly, another non-target site resistance mechanism for SDHI resistance was recently suggested to operate in some Z. tritici strains [32], but further research revealed that duplication of a SdhC paralog was responsible for this phenotype [50]. The presence of an additional SdhC paralog was found in several other fungi, including Fusarium graminearum and Alternaria alternata, but multiple copies have not been predicted for Magnaporthe oryzae.
The identification of multiple MDRP phenotypes associated with DMI resistance in our study warrants further research aimed at revealing the mechanisms of multidrug resistance in the wheat blast pathogen, including efflux pumps overexpression via transcriptomic analyses of differentially expressed genes; identification and discovery of mutations associated with changes in promoter regions or transcription factors of efflux transporters associated with multidrug resistance.
If allelic variation in the promoter regions, in the transcription factors or even in the coding region of the transporter genes could be unequivocally associated with MDR phenotypes, these alleles could be converted into molecular markers for monitoring, in real time, the spread of different MDR lineages of the pathogen in airborne inoculum [51,52,53]. This would foster the adoption of rational and sustainable anti-resistance strategies to manage wheat blast, using real time spatial and temporal disease surveillance tools based on the molecular detection of resistant inoculum of the pathogen in air samples from wheat fields. The adoption of such a smart, real-time anti-resistance strategy would minimize unneeded sprays and guide the choice of fungicides that are still effective when MDR is detected.
Future studies could be conducted by testing the role of specific transporter inhibitors such as BLT-4 [N-(2-methoxyphenyl)-N′-2-naphthalenyl-urea, ≈ INF271, a MFS transporter type inhibitor] and verapamil hydrochloride (for ABC transporter type inhibition) [16,54,55,56,57]. The main objective would be to assess if these two superfamily-distinct transporter inhibitors could interfere with the ability of PoTl strains for MDR resistance. One would look at any phenotypic changes induced by BLT-4 and verapamil hydrochloride in the fungal strain’s resistance responses to DMI and SDHI fungicides (i.e., altering the PoTl strain’s distinct MDRPs phenotypic patterns for MDR and restoring fungicide sensitivity).
Over the past few years, strategies have been prospected to combat MDR yeasts and bacterial human pathogens, as well as multi-resistant cancer cells, based on the concomitant administration of efflux pump exporters inhibitors using chemicals in synergism with azoles and other chemo drugs to restore their efficacy [58,59,60]. Perhaps this strategy could also be applied for managing MDR plant pathogens in the agroecosystem. Further studies could also be carried out in planta to assess whether the use of efflux pump inhibitors in association with DMI or SDHI fungicides would have an additive effect, restoring the efficacy of the fungicides for wheat blast control.

Author Contributions

Conceptualization, P.C.C.; methodology, P.C.C., S.I.M. and S.N.C.V.; software, P.C.C., S.N.C.V., S.I.M. and P.E.T.; validation, P.C.C. and B.F.; formal analysis, S.I.M., S.N.C.V. and P.C.C. and P.E.T.; investigation, A.G.d.S., F.G.A.J., L.D.K., S.N.C.V., S.I.M., T.C.S. and T.Y.K.d.O.; resources, P.C.C., A.A.d.P.C., B.F. and R.P.L.J.; data curation, S.I.M., S.N.C.V. and P.C.C.; writing—original draft preparation, S.I.M., S.N.C.V., P.C.C. and B.F.; writing—review and editing, P.C.C., B.F., A.A.d.P.C. and R.P.L.J.; visualization, P.C.C. and B.F.; supervision, P.C.C.; project administration, P.C.C., A.A.d.P.C., B.F. and R.P.L.J.; funding acquisition, P.C.C., A.A.d.P.C., B.F. and R.P.L.J. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the São Paulo Research Foundation, Brazil (FAPESP) through regular research grants to P.C.C. (2020/07611-9) in partnership with Fundação Araucária (F.A., Paraná, Brazil) to A.A.d.P.C. (FPS2020011000057); a Newton Fund grant from the Biotechnology and Biological Sciences Research Council (BBSRC) and UK Research and Innovation (UKRI), with FAPESP as partner institution in Brazil to P.C.C. (2017/50456-1; 2018/21197-0) and to S.I.M. (postdoctoral scholarship 2019/12509-1); a Brazilian National Research Council (CNPq) Grant/Award number: Pq-1D 313825/2018-1; Coordination for the Improvement of Higher Level Personnel (CAPES), Grant/Award Numbers: CAPES/AUXPE Program 88881.593505/2020-01—UNESP Ilha Solteira Campus, CAPES PrInt/UNESP, CAPES studentship Program 001). B.F. received funding from the Newton Fund through grant BB/S018867/2 awarded by BBSRC under the BBSRC-FAPESP AMR and Insecticide Pest Resistance in Livestock and Agriculture Programme. T.Y.K.d.O. and F.G.A.J. were supported by a M.Sc. research scholarship from CAPES. S.N.C.V., A.G.d.S., T.C.S. and L.D.K. are supported by Ph.D scholarships from CAPES. The article processing charges were supported mostly by São Paulo State University’s Vice-Presidency for Research (PROPE) special grant for publication (Public Call 10/2022) and partially by funds from the Paraná Rural Development Institute, IDR—Paraná/IAPAR.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

The Brazilian Ministry of Environment/National System for the Management of Genetic Heritage and Associated Traditional Knowledge—SisGen, issued the Certificates # A59E163 and A100786 authorizing the scientific activities associated with the collection of botanical and fungal material from the wheat agroecosystem in the Cerrado´s biome and access the genetic diversity of Pyricularia species.

Data Availability Statement

The phenotypic data on EC50 values for sensitivity to the antifungal efflux pump substrates tolnaftate (TOL), cycloheximide (CHX), rhodamine 6G (RH6G) and triphenyltin chloride (TPCL) presented in this study are available upon request to the corresponding author. The data are not publicly available due to the authors’ option.

Conflicts of Interest

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

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Agronomy 12 02068 g001 550
Figure 1. Contrasting in vitro relative growth of Pyricularia oryzae Triticum and Oryza lineages in different efflux pump substrates in PD broth on a microplate assay. (A). Triphenyltin chloride (TPCL)-resistant strain 18SPK6 from P. oryzae Triticum lineage (PoTl), and the sensitive strain 421 from P. oryzae Oryza lineage (PoOl). (B). Tolnaftate (TOL)-resistant PoTl 12.1.130 and the sensitive strain PoOl 421. (C). Rhodamine 6G (RH6G)-resistant strain PoTl 12.1.299 and the sensitive strain PoOl 421. (D). Cycloheximide (CHX)-resistant strain PoTl 18MGF23 and the sensitive strain PoOl 704. Bars contain the sample median and outer quantiles at 95% confidence interval (the 2.5th and the 97.5th percentiles as the lower and the upper confidence limits). Data points are distributed along the bar.
Figure 1. Contrasting in vitro relative growth of Pyricularia oryzae Triticum and Oryza lineages in different efflux pump substrates in PD broth on a microplate assay. (A). Triphenyltin chloride (TPCL)-resistant strain 18SPK6 from P. oryzae Triticum lineage (PoTl), and the sensitive strain 421 from P. oryzae Oryza lineage (PoOl). (B). Tolnaftate (TOL)-resistant PoTl 12.1.130 and the sensitive strain PoOl 421. (C). Rhodamine 6G (RH6G)-resistant strain PoTl 12.1.299 and the sensitive strain PoOl 421. (D). Cycloheximide (CHX)-resistant strain PoTl 18MGF23 and the sensitive strain PoOl 704. Bars contain the sample median and outer quantiles at 95% confidence interval (the 2.5th and the 97.5th percentiles as the lower and the upper confidence limits). Data points are distributed along the bar.
Agronomy 12 02068 g001
Agronomy 12 02068 g002 550
Figure 2. Pearson correlation analysis among the EC50 values for the efflux pump substrates tolnaftate (TOL), cycloheximide (CHX), rhodamine 6G (RH6G) and triphenyltin chloride (TPCL) and the site-specific fungicides tebuconazole (DMI) and fluxapyroxad (SDHI) determined for Pyricularia oryzae Triticum and Oryza lineages and P. grisea. Diagonal frames represent the distribution of the EC50 values for each chemical; frames below, the bivariate dispersion and adjusted lines; frames above, the correlations values and the significance levels. Significance of the correlation was determined by t tests. p values were represented by: *** = 0.001; ** = 0.01; • = 0.1.
Figure 2. Pearson correlation analysis among the EC50 values for the efflux pump substrates tolnaftate (TOL), cycloheximide (CHX), rhodamine 6G (RH6G) and triphenyltin chloride (TPCL) and the site-specific fungicides tebuconazole (DMI) and fluxapyroxad (SDHI) determined for Pyricularia oryzae Triticum and Oryza lineages and P. grisea. Diagonal frames represent the distribution of the EC50 values for each chemical; frames below, the bivariate dispersion and adjusted lines; frames above, the correlations values and the significance levels. Significance of the correlation was determined by t tests. p values were represented by: *** = 0.001; ** = 0.01; • = 0.1.
Agronomy 12 02068 g002
Table
Table 1. Analysis of variance of the effect of strains on the EC50 values for efflux pump substrates.
Table 1. Analysis of variance of the effect of strains on the EC50 values for efflux pump substrates.
Source of VariationdfTriphenyltin ChlorideTolnaftateRhodamine 6GCycloheximide
Mean SquareF ValuesMean SquareF ValuesMean SquareF ValuesMean SquareF Values
Strain (S)200.080253.923 **5,04343.241 **2.9466.325 **0.557520.851 **
Block30.00211.431 NS0.09650.828 NS0.12370.265 NS0.08813.295 NS
Residuals1220.0015 0,1166 0.4659 0.0267
** Significance by the F test at p < 0.01; NS non-significant. The experiment was repeated once and a joint analysis of the data was performed, since no interaction strain vs. experiment was detected in the individual analyses.
Table
Table 2. Field isolates of Pyricularia spp. sampled in Brazil in 2012 and 2018 and respective resistance phenotype classes to three major fungicide classes and sensitivity to efflux pump substrates and respective resistance factors.
Table 2. Field isolates of Pyricularia spp. sampled in Brazil in 2012 and 2018 and respective resistance phenotype classes to three major fungicide classes and sensitivity to efflux pump substrates and respective resistance factors.
a Species and Isolatesb Previously Determined Fungicide Resistance Phenotypef,g,h,i Mean EC50 (μg·mL−1), Standard Deviation and RF j MDR Phenotype
c QoI Azoxystrobind DMI Tebuconazolee SDHI
Fluxapyroxad		Triphenyltin Chloride (TPCL) RF TPCLTolnaftate
(TOL)		 RF TOLRhodamine 6G
(RH6G)		 RF RH6GCycloheximide
(CHX)		 RF CHX
Pyricularia oryzae Triticum lineage—PoTl
12.1.015SRRS0.030 (0.01)d3.50.25 (0.1)d0.731.01 (0.9)b290.10 (0.08)d0.83MDRP1
12.1.045iRRRS0.036 (0.01)d4.20.36 (0.1)d1.11.26 (0.41)b370.13 (0.09)d1.1MDRP1
12.1.037RRRS0.04 (0.002)d4.70.31 (0.3)d0.911.22 (0.8)b350.09 (0.07)d0.75MDRP1
18MGH25RMRRS0.06 (0.03)d7.10.62 (0.33)d1.80.80 (0.54)c230.18 (0.11)d1.5MDRP2
18MGH11RRHR0.04 (0.005)d4.70.25 (0.07)d0.740.47 (0.27)c130.17 (0.14)d1.4MDRP1
12.1.005RRS0.04 (0.03)d4.70.55 (0.29)d1.61.76 (1.1)a500.18 (01.5)d1.5MDRP1
12.1.183RRMR0.16 (0.06)b190.87 (0.49)c2.61.07 (0.83)b310.42 (0.15)c3.5MDRP2
18MGH19RRRS0.12 (0.02)c141.02 (0.30)c3.00.73 (1.08)c210.27 (0.45)c2.3MDRP2
18SPK6RRRS0.31 (0.09)a361.33 (0.68)b3.91.77 (0.80)a510.15 (0.05)d1.3MDRP2
12.1.299RRMR0.17 (0.04)b200.79 (0.57)c2.32.13 (0.65)a610.11 (0.09)d1.1MDRP2
12.1.312RRMR0.19 (0.08)b220.89 (0.64)c2.61.03 (0.34)b290.11 (0.04)d0.92MDRP 2
18MGF23RRMR0.15 (0.03)b180.31 (0.12)d0.911.35 (0.29)b391.24 (0.53)a10MDRP4
18SPC10 bRRHR0.31 (0.04)a360.50 (0.31)d1.51.25 (1.28)b360.13 (0.03)d1.1MDRP2
12.1.165SRS0.27 (0.05)a320.36 (0.09)d1.11.47 (1.00)b420.16 (0.06)d1.3MDRP2
12.1.130SHRRS0.04 (0.002)d4.74.40 (2.2)a131.87 (0.6)a530.11 (0.03)d0.92MDRP3
18PRH9RRS0.03 (0.004)d3.50.87 (0.45)c2.60.50 (0.6)c140.12 (0.03)d1.0MDRP1
Pyricularia oryzae Oryza lineage—PoO
656SSS0.013 (0.007)d1.530.45 (0.28)d1.320.06 (0.05)d1.710.12 (0.1)d1.0-
421SSS0.004 (0.001)d0.470.23 (0.08)d0.680.01 (0.01)d0.290.80 (0.34)b6.7-
674SSRS0.033 (0.03)d3.880.42 (0.33)d1.240.25 (0.17)c7.140.09 (0.09)d0.75-
704SSMR0.004 (0.001)d0.470.28 (0.09)d0.820.05 (0.03)d1.430.09 (0.06)d0.75-
Pyricularia grisea—Pg
363RSS0.04 (0.004)d4.710.26 (0.08)d0.761.67 (1.4)a480.11 (0.05)d0.92-
a The PoTl strain coding indicates the respective sampling year: ‘12’ for 2012 populations and ‘18’ for 2018 populations; PoOl and Pg isolates were from 2012 populations. b Sensitivity phenotype classes: S = sensitive, RS = reduced sensitivity, MR = moderately resistant, R = resistant, HR = highly resistant. c According to Castroagudín et al. [6]: R isolates grew at the discriminatory dose of 10 μg·mL−1. d According to Poloni et al. [9]: S, EC50 = 0.007–0.07 μg·mL−1; MR, EC50 = 0.62–0.98 μg·mL−1; R, EC50 = 1.03–1.44 μg·mL−1; HR ≥ 1.62–1.70 μg·mL−1. e According to Vicentini et al. [10]: S, EC50 = 1.15–2.94 μg·mL−1; RS, EC50 = 3.30–19.31 μg·mL−1; MR, EC50 = 21.83–43.67 μg·mL−1; HR ≥ 50–100 μg·mL−1. f Effective concentration to inhibit fungal growth in 50% (EC50) using mycelial fragment method evaluation by absorbance at 620 nm in microplates. g Means followed by the same letters in the column did not differ by the Scott-Knott test (at p ≤ 0.05). Standard deviation in parenthesis. h Resistance factor (RF) indicates the ratio of the strain’s EC50 tested to the mean average EC50 value for strains PoOl 421 and 656 for each antifungal- isolate combination, with exception for cycloheximide where only the EC50 value of PoOl 656 was used due to insensitivity of strain PoOl 421. i RF within each substrate: dark purple = very high, orange = high, light purple = medium. j Multidrug-resistance phenotype of PoTl strains (MDRP) classification according to the combination of resistance to different efflux pump substrates and resistance to tebuconazole (DMI) and/or fluxapyroxad (SDHI).
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Vicentini, S.N.C.; Moreira, S.I.; da Silva, A.G.; de Oliveira, T.Y.K.; Silva, T.C.; Assis Junior, F.G.; Krug, L.D.; de Paiva Custódio, A.A.; Leite Júnior, R.P.; Teodoro, P.E.; et al. Efflux Pumps and Multidrug-Resistance in Pyricularia oryzae Triticum Lineage. Agronomy 2022, 12, 2068. https://doi.org/10.3390/agronomy12092068

AMA Style

Vicentini SNC, Moreira SI, da Silva AG, de Oliveira TYK, Silva TC, Assis Junior FG, Krug LD, de Paiva Custódio AA, Leite Júnior RP, Teodoro PE, et al. Efflux Pumps and Multidrug-Resistance in Pyricularia oryzae Triticum Lineage. Agronomy. 2022; 12(9):2068. https://doi.org/10.3390/agronomy12092068

Chicago/Turabian Style

Vicentini, Samara Nunes Campos, Silvino Intra Moreira, Abimael Gomes da Silva, Tamiris Yoshie Kiyama de Oliveira, Tatiane Carla Silva, Fabio Gomes Assis Junior, Loane Dantas Krug, Adriano Augusto de Paiva Custódio, Rui Pereira Leite Júnior, Paulo Eduardo Teodoro, and et al. 2022. "Efflux Pumps and Multidrug-Resistance in Pyricularia oryzae Triticum Lineage" Agronomy 12, no. 9: 2068. https://doi.org/10.3390/agronomy12092068

APA Style

Vicentini, S. N. C., Moreira, S. I., da Silva, A. G., de Oliveira, T. Y. K., Silva, T. C., Assis Junior, F. G., Krug, L. D., de Paiva Custódio, A. A., Leite Júnior, R. P., Teodoro, P. E., Fraaije, B., & Ceresini, P. C. (2022). Efflux Pumps and Multidrug-Resistance in Pyricularia oryzae Triticum Lineage. Agronomy, 12(9), 2068. https://doi.org/10.3390/agronomy12092068

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