Resistance to Triazole Fungicides in Pyricularia Species is Associated with Invasive Plants from Wheat Fields in Brazil

Resistance to triazole fungicides in Pyricularia species is associated with invasive plants from wheat fields in Brazil. Acta Scientiarum. Resistance to triazole fungicides in Pyricularia species Abstract Triazole fungicides have not been effective for managing the wheat blast disease in Brazil. A broad analysis across six geographical populations of Pyricularia graminis-tritici in central-southern Brazil indicated a high level of resistance to triazole fungicides. Since P. graminis-tritici is also associated with others poaceous species, here, we analyzed whether triazole-resistant isolates of the blast pathogen could be recovered from other poaceous hosts that are invasive of sprayed wheat fields. In addition to P. graminis-tritici (Pygt), we also evaluated the levels of sensitivity of three other grass-associated P. ( Pu ) . Resistance to the triazole fungicides tebuconazole and epoxiconazole was assessed phenotypically based on EC 50 values and molecularly by analysis of the presence of mutations in the CYP51A gene, which encodes for the target enzyme 14-alpha-demethylase. We detected triazole-resistant Pyricularia spp. ( Pg , Pp , Pu and Pygt ) that is associated with Avena sativa , Cenchrus echinatus , Chloris distichophylla , Cynodon sp., Digitaria horizontalis , D. sanguinalis , Panicum maximum or Urochloa spp. The major outcome from our study was the evidence that invasive poaceous species from wheat fields could be an important source of triazole resistant fungal inoculum for the initial phases of the wheat blast epidemics.


Introduction
Since the late 1980's, wheat blast has been considered a major disease, causing high yield losses on crops from central-southern Brazil (Maciel, 2011;Maciel et al., 2014).After its first report in 1986 in northern Paraná State, Brazil (Igarashi, 1986), it has rapidly spread to all of the wheat cropping areas of the country as well as to Argentina, Bolivia, and Paraguay (Maciel, 2011).Initially restricted to South America, wheat blast was recently introduced in Bangladesh in Southeastern Asia (Callaway, 2016;Islam et al., 2016).
Management of wheat blast disease is considered particularly difficult due to the inexistence of durable varietal resistance and the lack of effective systemic fungicides (Maciel et al., 2014;Pagani, Dianese, & Café Filho, 2014;Castroagudin et al., 2015).Despite their low efficacy for controlling blast disease on wheat ears, systemic fungicides such as triazoles have been extensively and widely used in Brazilian wheat fields since the 1990's for managing other fungal diseases, including leaf rust, powdery mildew, leaf spots and gibberella diseases (Navarini & Balardin, 2012;Tormen et al., 2013;Debona, Favera, Corte, Domingues, & Balardin, 2009).
The emergence of fungicide resistance could be one of the main causes of the low efficacy of triazole (Ceresini et al., 2018).A strong selection pressure resulting from several years of extensive and frequent applications of triazoles for disease control may have triggered the emergence of resistant pathogen populations (Lucas, Hawkins, & Fraaije, 2015).This scenario of intensive triazole usage leading to the emergence of resistance and reduced fungicide efficacy has been reported in Europe, South America, and Asia for many plant pathogens associated with cereal crops such as Erysiphe graminis on barley and wheat (Buchenauer & Hellwald, 1985) and Mycosphaerella graminicola (Brunner, Stefanato, & McDonald, 2008) and Parastagonospora nodorum on wheat (Pereira, McDonald, & Brunner, 2016).
Resistance to triazoles may be directly related to (1) mutations in the CYP51 gene that encode the target protein resulting in decreased protein affinity for the inhibitors; (2) overexpression of the CYP51 gene, and (3) increased efflux of toxic compounds out of the fungal cell due to overexpression of the gene encoding membrane transport proteins.A combination of these mechanisms is also possible (Cools & Fraaije, 2013).Mutations in the paralog A of the CYP51 gene (CYP51A) were considered the primary cause for the reduction in sensitivity to triazoles in fungi with multiple CYP51 genes, such as the wheat head blight pathogen Fusarium graminearum (Jiang, Liu, Yin, & Ma, 2011;Fan et al., 2013), the rice blast pathogen Pyricularia oryzae (Yan et al., 2011), and the wheat blast pathogen P. graminis-tritici (Ceresini et al., 2018).In fact, widespread distribution of resistance to the triazole fungicides epoxiconazole and tebuconazole has been reported in populations of P. graminis-tritici from several wheat fields in central-southern Brazil (Ceresini et al., 2018).
Since P. graminis-tritici can also be associated with others poaceous species (Castroagudin et al., 2016), the main objective of our study was to test whether triazole-resistant isolates of the wheat blast pathogen could be recovered from other poaceous hosts that are invasive of triazole -sprayed wheat fields.Since three other blast pathogens (P.grisea, P. pennisetigena, and P. urashimae) were obtained from the sampling of these invasive poaceous hosts in our study, we tested the additional hypothesis that triazole spraying on wheat fields has contributed to and selected for resistance in non -target Pyricularia species.
Up until now, there has been no report of the occurrence of triazole resistance in populations of blast pathogens associated with other poaceous hosts that are invasive of wheat fields in Brazil.Thereby, we evaluated the levels of sensitivity of the blast pathogens P. graminis tritici, P. grisea, P. pennisetigena, and P. urashimae to the triazole fungicides epoxiconazole and tebuconazole.The levels of triazole sensitivity were determined based on the individual EC50 values (the effective concentration that inhibits 50% of mycelial growth).We also analyzed the CYP51A gene for the presence of particular mutations that could be correlated with triazole resistance.Finally, a reticulate phylogeny of the CYP51A gene was built and examined to describe the evolutionary relationships among haplotypes of the Pyricularia species.

Material and methods
Thirty-two isolates of Pyricularia ssp.were used in this study, of which 28 were from blast-diseased poaceous species that are invasive to wheat fields and four from wheat blast.These isolates comprised four Pyricularia species: P. grisea (Pg, N = 4), P. pennisetigena (Pp, N = 4), P. urashimae (Pu, N = 4) and P. graminis-tritici (Pygt, N = 20).These isolates were obtained in 2012 by sampling diseased plants using transect system in Paraná (PR) and Mato Grosso do Sul (MS) and later identified at the specie s level (Castroagudin et al., 2016;Crous et al., 2016;Reges et al., 2016).In addition, seven isolates of P. oryzae (Po), sampled from rice fields in Goiás (GO) and Tocantins (TO) in 2007, were included as sensitive standards (Table 1).
For fungal DNA extraction, the mycelial mass of thirty-nine isolates of Pyricularia spp. was obtained by cultivation in PD broth (potato-dextrose, Himedia, Mumbai, MA, India) for seven days at 24°C and 150 rpm.The DNA was extracted with the GenElute Plant Genomic DNA Miniprep kit (Sigma -Aldrich, USA) according to the manufacturer's recommendations and quantified using a spectrophotometer NanoDrop® 2000c (Thermo Fisher Scientific, USA).
Seven primers were designed to ensure amplification of the CYP51A gene for all five Pyricularia species that were included in our study (Table 2).For each species, three distinct primer combinations were used in PCR reactions for complete coverage of the CYP51A gene (Additional file 1, Table 2).Polymerase chain reaction (PCR) was performed in a ProFlex thermal cycler (Applied Biosystems, USA) with a final volume of reaction of 25 µL containing ultrapure distilled water, 50 ng of total fungal DNA, 0.3 µM each primer, 0.2 mM dNTP, 2 mM MgCl2, 2.5 µL of 10X buffer and 1 U of Taq DNA polymerase (Sigma-Aldrich, USA).The following cycling conditions were used: initial denaturation at 95°C for 7 min.;35 cycles of 95°C for 1 min., 52°C as annealing temperature for 1 min., and 72°C for 1 min.;and final extension at 72°C for 7 min.for Pg, Pp, Pu, and Pygt, while for Po, the annealing temperature was set at 55°C.Amplifications of the DNA fragments were checked on a 1% agarose gel.The sequencing reactions were performed at Macrogen Inc., Seoul, South Kor ea, using an ABI 3700 DNA analyzer.To obtain total coverage of the CYP51A gene (1551 bp), three sequencing reactions using primers described in Additional file 1 and Table 2 were performed for each isolate, and the fragments obtained were aligned to generate a consensus sequence.The consensus DNA sequences for each isolate were aligned and analyzed using the software Geneious R v. 9.0.5 (Biomatters, Auckland, New Zealand).The complete sequence of CYP51A for Pp, Pu, Pygt, and Po was 1551 bp in length, while for Pg, it was 1420 bp (~92% of gene coverage).
Haplotype frequencies were determined using the program DnaSP version 5.10.1 (Rozas, 2009).The CYP51A gene sequences were checked for synonymous and non-synonymous mutations using as reference the CYP51A gene sequence from Po isolate 622 (with sensitive phenotype for both tebuconazole and epoxiconazole).
The phylogenetic relationships among distinct CYP51A haplotypes were determined by reconstructing a reticulate phylogeny using the parsimonious statistical method implemented in the program TCS version 1.21 (Clement, Posada, & Crandall, 2000).We also build an UPGMA phylogenetic tree using the Geneious R tool Tree Builder, assuming the evolutionary model HKY.The internode support for the branches was tested by bootstrap with 1,000 data resampling .

Results and discussion
Increasing concentrations of the DMI fungicides tebuconazole and epoxiconazole resulted in mycelial growth reduction for all isolates of Pyricularia spp.from different poaceous species (Figure 1; Additional file 2).There were also differences among isolates within species, allowing for the discrimination between extreme DMI-resistant and DMI-sensitive phenotypes in four out of the five Pyricularia spp.examined (Pg, Pp, Pu, and Pygt; Additional file 2).The mean species effect was significant at p ≤ 0.001 for both tebuconazole and epoxiconazole, and the EC50 values were significantly different among Pyricularia species (Scott-Knott at p ≤ 0.05) (Figure 2).Pygt and Pp were highly resistant to tebuconazole, showing the highest EC50 values (EC50 = 1.438 and = 1.421 µg mL -1 , respectively), and significantly different from the other species ( p ≤ 0.05).Pu and Pg where also resistant to tebuconazole, but with intermediate EC 50 values varying from 0.771 to 1.074 µg mL -1 , respectively.In contrast, Po was highly sensitive to tebuconazole, with a very low mean EC50 = 0.036 µg mL -1 , which was significantly lower than that of all the other Pyricularia species examined (Figure 2).
From a total of 13 mutations detected along the CYP51A gene from Pp, Pu and Pygt, one was nonsynonymous, while 12 were synonymous.The single nonsynonymous mutation was found at residue 158 of the CYP51A gene with a resulting amino acid change from arginine to lysine R158K (Table 3).
For Pg, in contrast, in addition to the mutation resulting in the R158K substitution, another 51 nonsynonymous mutations were detected.The occurrence of these 52 mutations was possibly related to the phylogenetic distance between Pg and the group that includes the sister species Pp, Pu, and Pygt (Figure 3).In fact, the phylogenetic tree for the CYP51A gene evidenced that the species Pp, Pu, and Pygt shared the identical haplotype H1, which was closely related to H7 but distinct from Po, all with high bootstrap support.In contrast, the clade containing Pp, Pu, Pygt, and Po was only 80.9% similar to Pg (Additional file 3).The reconstruction of a reticulate phylogeny also allowed for the depiction of nucleotide variation within the CYP51A gene, the frequency of occurrence of the haplotypes detected, and their relationships (Figure 4).Four distinct haplotypes were detected: H6 -DMI sensitive, associated exclusively with Po (MF381150) (N = 7); H1 -DMI resistant, which was the most frequent among the haplotypes (N = 18), associated with Pp (MF381153), Pu (MF381152) and Pygt (MF381151), and distinct from H6 by eleven mutational steps; H7 -DMI resistant, which was found only once (i.e., a singleton) in the Pygt isolate 12.0.145(MF381154); and the haplotype H8 -DMI resistant, also detected only once in Pg (MF381155), which was separated from H6 by 48 mutational steps (Figure 4).The most frequent haplotype, H1, and haplotype H7 were differentiated by only one synonymous mutation (C756T).
The major outcome of our study was the evidence that invasive poaceous species from wheat fields could be an important DMI-resistant fungal inoculum source for the initial phases of a wheat blast epidemic (Urashima & Kato, 1998).Additionally, the invasive poaceous species that is closer to the fungicide-sprayed wheat fields could have an important role as a DMI-resistant inoculum reservoir between cropping seasons.Three CYP51A haplotypes (H1, H7 and H8) associated with resistance to DMI fungicides were detected among the Pyricularia species that were sampled from several invasive plant species in wheat fields.Castroagudin et al. (2015), who surveyed Pyricularia spp.associated with invasive plants from wheat fields, also detected two of the most common cytB haplotypes (H1 and H3) containing the G143A mutation that confers resistance to the QoI fungicide azoxystrobin.The H1-QoI resistant haplotype was detected in 48% of the isolates from invasive plant species, mostly from signal grass (Urochloa spp.) and weeping finger grass (Chloris distichophylla).
In terms of levels of resistance, with the exception of the rice blast fungus P. oryzae, which was used as our sensitive standard, all of the other four Pyricularia species (Pygt, Pg, Pp, and Pu) associated with invasive Poaceae were resistant to DMI fungicides.
For epoxiconazole, the mean EC50 for the four Pyricularia species was 0.33 µg mL -1 (with a minimum of 0.10 and a maximum of 0.95 µg mL -1 ).In comparison with the same wheat or other cereals' pathogens, the following epoxiconazole EC50 were described: Mycosphaerella graminicola [maximum EC50 = 0.05 µg mL -1 (Stammler & Semar, 2011), Rhynchosporium commune [maximum EC50 = 0.23 µg mL -1 (Hawkins et al., 2014)] and Zymoseptoria tritici [maximum EC50 = 0.48 µg mL -1 (Cools & Fraaije, 2013).Based on the labeled doses of 75 g epoxiconazole ha -1 and 150 g tebuconazole ha -1 recommended for fungicide sprays in Brazilian wheat fields by the Brazilian Ministry of Agriculture, Livestock and Food Supply -MAPA (http://agrofit.agricultura.gov.br/agrofit_cons/principal_agrofit_cons), and using the method proposed by Castroagudin et al. (2015) considering the average wheat plant height at heading stage of approximately 0.82 meters, we estimated that epoxiconazole and tebuconazole are sprayed at the concentrations of 0.0055 µg mL -1 and 0.0180 µg mL -1 per hectare of wheat fields, respectively.Comparing these resulting field concentrations with the mean EC50 values that were estimated, we concluded that, on average, all four Pyricularia species from invasive grasses resisted at 60 to 65 times higher field doses of epoxiconazole or tebuconazole, respectively.
An R158K point mutation in the CYP51A gene that was conserved in four Pyricularia species (Pg, Pp, Pu, and Pygt) but absent in the reference species for DMI sensitivity (Po) may be related to resistance to tebuconazole and epoxiconazole.
In a countrywide population-based study in which 178 Pygt isolates were sampled from seven wheat fields in Brazil, Ceresini et al. (2018) also described the predominance of the R158K mutation in all four DMI-resistant haplotypes detected (H1, H2, H3, and H4).The predominant CYP51A haplotype associated with Pp, Pu and Pygt from invasive species was identical to the H1 haplotype described by (Ceresini et al. 2018), which was the most commonly found in all seven wheat blast populations (N = 175).
However, as reported for few other fungal plant pathogens, in addition to target site mutations found in the CYP51A gene of Pg, Pp, Pu, and Pygt, resistance to DMI fungicides could also be related to other mechanisms of quantitative nature, such as increase in ABC transporter efflux (Nakaune et al., 1998) and overexpression of the CYP51A gene (Ma et al., 2005;Coleman & Mylonakis, 2009;Abou Ammar et al., 2013).
Due to the quantitative and polygenic nature of the resistance attributed to DMI fungicides, the resistance to DMI fungicides found in all four Pyricularia spp.from Poaceae species that are invasive to wheat fields may be a result of slow and gradual selective pressure exerted on the pathogen populations due to long-term use of DMI fungicides at high dosages (Deising, Reimann, & Pascholati, 2008;Lucas et al., 2015).
To avoid the intensification of this scenario over the next few years, the adoption of anti-resistance management strategies is urgently needed.To decrease the selective pressure towards resistant pathogen populations, such strategies would include rotations of fungicides with different modes of action (Milgroom & Fry, 1988) and adoption of mixtures of single-target-site, high-risk fungicides with multiple-target-site, low-risk fungicides (Lucas et al., 2015).

Conclusion
All four Pyricularia species (Pygt, Pg, Pp, and Pu) associated with invasive Poaceae were resistant to DMI fungicides.
Several invasive poaceous species adjacent to sprayed wheat fields constitute an important inoculum reservoir of DMI-resistant Pyricularia spp.for the initial phases of the blast epidemic, especially for wheat blast.fellowship from CAPES.We thank CAPES for sponsoring the establishment of the "Centro de Diversidade Gene tica no Agroecossistema" (Pro-equipamentos 775202/2012).

Figure 2 .
Figure 2. Boxplot representing the variation in EC50 values to DMI fungicides by isolates of Pyricularia spp.The mean EC50 values for isolates of each Pyricularia species with the CYP51A gene sequenced were indicated by a red line a .a A complete randomized experimental design with six repetitions per fungal isolate of each species was used.The experiment was replicated once.Data from the two experiments were combined for the variance analyses because there were no significant differences between experiments (Ftebuconazole experiments = 1.945NS , p = 0.1639; Fepoxiconazole experiments = 3.166 NS , p = 0.0759) and the ranking of species based on their EC50 values was consistent across experiments, indicating no significant interaction.The species effect was significant for both fungicides (Ftebuconazole species = 255.27*** , p < 0.001; Fepoxiconazole species = 159.42*** , p < 0.001).Five Pyricularia species were compared: P. grisea (Pg, N = 4 isolates), P. pennisetigena (Pp, N = 4) P. urashimae (Pu, N =4), P. graminis-tritici (Pygt, N = 20) and P. oryzae (Po, N = 7).Boxplots followed by the same capital letters indicated no significant differences between species in EC50 values (Scott-Knott test at p < 0.05).

Figure 4 .
Figure 4. Network of haplotypes of the CYP51A gene from Pyricularia spp.The area of each circle is proportional to the number of isolates sampled from each haplotype.The lines between one circle and another represent the mutational steps between haplotypes.

Table 1 .
Description of isolates of Pyricularia species associated with blast disease on invasive poaceous species from wheat fields that were used for assessing DMI fungicide resistance a .
a Seven isolates of the rice blast pathogen (Po) were included as DMI-sensitive references, while four Pygt isolates from wheat blast were included as DMIresistant isolates.

Table 2 .
Description of primers used for amplification and sequencing of CYP51A genes from Pyricularia species associated with invasive plants and rice in Brazil.
a * Indicates primer used for polymerase chain reaction amplification.

Table 3 .
Description of mutations found in the sequences of the CYP51A gene of Pyricularia spp.