Status of Fungicide Resistance and Physiological Characterization of Tebuconazole Resistance in Rhizocotonia solani in Sichuan Province, China

The resistance prevalence of chemical fungicides has caused increasingly serious agro-ecological environmental problems. However, there are few previous reports about resistance to succinate dehydrogenase (SDHI) or sterol demethylation inhibitor (DMI) in Rhizoctonia solani, one of the main agro-diseases. In this study, the fungicide resistance of 122 R. solani isolates in Sichuan Province was monitored by the mycelial growth rate method. Results showed that all isolates were susceptible to hexaconazole and most isolates were susceptible to thifluzamide, except for the field isolate MSRS-2-7 due to a moderate resistance to thifluzamide (16.43-fold resistance ratio, RR), compared to the sensitivity baseline of thifluzamide (0.042 μg/mL EC50 values). On the contrary, many isolates showed moderate or high resistance to tebuconazole (10.59- to 60.78-fold RR), reaching EC50 values of 0.54~3.10 μg/mL, especially for a highly resistant isolate LZHJ-1-8 displaying moderate resistance to epoxiconazole (35.40-fold RR due to a 3.54 μg/mL EC50 value). The fitness determination found that the tebuconazole-resistant isolates showed higher fitness cost with these characteristics, including a lower growth rate, higher relative electric conductivity, an increased ability to tolerate tebuconazole, and high osmotic pressure. Four new mutations of cytochrome P450 sterol 14α-demethylase (CYP51), namely, S94A, N406S, H793R, and L750P, which is the target for DMI fungicides, was found in the tebuconazole-resistant isolates. Furthermore, the lowest binding energy with tebuconazole was also found in the LZHJ-1-8 isolate possessing all the mutations through analyses with Discovery Studio software. Therefore, these new mutation sites of CYP51 may be linked to the resistance against tebuconazole, and its application for controlling R. solani should be restricted in some areas.


Introduction
Rice sheath blight, caused by Rhizoctonia solani Kuhn AG1-1A [teleomorph Thanatephorus cucumeris (A. B. Frank) Donk], is a common disease in rice and mostly occurs under high temperature and humidity. The disease can produce incomplete grains, more scum, and even lodging [1]. It can cause a yield loss of 20-50% in susceptible rice cultivation areas and shows an increasing trend due to the irrational use of nitrogen fertilizer and change in global climate conditions [2]. The measures to control R. solani mainly include strengthening management of field fertilizer and water, reduction in bacterial sources, and cultivation of resistant varieties [3]. However, the application of chemical fungicides is the most popular and effective control measure [4]. In rice production, the common chemical fungicides for controlling rice sheath blight consist of succinate dehydrogenase inhibitors

Determination of Resistance Frequency of R. solani
The minimum inhibitory concentration (MIC) was used to determine the resistance frequency of R. solani [19]. The MICs of four fungicides were based on their lowest inhibitory concentration [20] against an indoor susceptible isolate obtained from Southwest Crop Genetic Resource Discovery and Utilization Laboratory of Sichuan Agricultural University; the MICs of thifluzamide, hexaconazole, tebuconazole, and epoxiconazole were 5, 15, 20, and 10 µg/mL, respectively. The 122 isolates were placed on a PSA medium plate, previously stored in a refrigerator at 4 • C, and activated at a constant temperature of 28 • C for 36 h; then, mycelia with a 5 mm diameter were punched out with a hole punch, and inoculated on PSA medium containing the identified MICs. After culturing in the dark at 28 • C for 36 h, those isolates that could not grow normally were identified as susceptible isolates; on the contrary, the isolates that could grow normally were identified as resistant isolates, and the mycelia diameter was measured by the cross method [21]. The mycelia diameter grown on the drug-containing medium was marked A, and that on the drug-free medium was marked B. The occurrence frequency of susceptible and resistant isolates, and the mycelial growth inhibition rate, were calculated [22]. Mycelial growth inhibition rate (%) = [1 − (A − 5 mm)/(B − 5 mm)] × 100%

Sensitivity Baseline or Resistance Ratio of R. solani to the Tested Fungicides
According to the resistance frequency and mycelial growth inhibition rate results, some representatives from sensitive and resistant (with a least inhibition rate) isolates of R. solani to thifluzamide, hexaconazole, tebuconazole, and epoxiconazole were selected, and their EC 50 values against these four fungicides were determined by the mycelial growth rate method [23]. Each treatment was replicated three times. A mycelium with a diameter of 5 mm was inoculated into the center of the drug-containing medium, and the drugfree medium was used as a blank control. After culturing at 28 • C for 36 h, the mycelia diameter was measured by the cross method [21]. A regression equation was derived by correlating the log10 of inhibitor concentration and the probability value of the mycelial growth inhibition rate, while effective concentration for 50% inhibition rate (EC 50 ) was calculated from the regression equation [7]. Resistance ratio (RR) was obtained as the ratio of EC 50 value for resistant isolates to EC 50 value for sensitive isolates.

Fitness Determination of Tebuconazole-Resistant Isolates
Referring to the method reported by Dolores et al. [24], to test the osmotic sensitivity of the tebuconazole-sensitive or resistant isolates to glucose, mycelium plugs with 5 mm diameter were punched at the edge of the mycelia, and inoculated onto PDA medium containing 1%, 2%, 4%, and 8% glucose, after activation at 28 • C for 36 h. Then, the mycelia growth diameter was measured by the cross method, and each treatment was repeated 3 times.
To test the osmotic sensitivity of these isolates towards NaCl, mycelium plugs with a 5 mm diameter from the edge of 36 h activated mycelia were transferred into PSA medium containing mass concentration of 0, 1.25, 2.5, 5, 10, 20, 40, and 80 g/L NaCl. Each isolate was incubated at 28 • C for 36 h with three replicates. The growth diameters of mycelia on medium with different concentrations of NaCl were measured by the cross method [21].

Determination of Cell Membrane Permeability
The cell membrane relative permeability rate for tebuconazole-sensitive or resistant isolates was evaluated according to the described method [25] with some modifications. The activated mycelia were respectively inserted into the PDB medium, and shaken (120 r/min) for 3 d; then, the fresh mycelium was collected, and later washed with double-distilled sterile water and vacuum filtered. Then, 0.5 g of the fresh weight was put into a conical triangular flask, consisting of 0, 0.5, 1.0, 5.0, and 25.0 µg/mL tebuconazole diluted with double distilled sterile water. After shaking (120 r/min) in a constant temperature water bath at 28 • C for 0, 5, 10, 15, 30, 60, 90, 120, 180, 240, 300, 360, 420, and 540 min, the conductivity was measured using a DDS-11A meter, and the boiled dead mycelium was treated as a control. Each treatment was repeated 3 times.

Clone of CYP51 Gene from R. solani Isolates
Genomic DNA from mycelium of R. solani isolates were extracted using the EasyPure Plant Genomic DNA Kit (TransGen Biotech, Beijing, China) according to the manufacturer's recommendations [26]. The PCR primers (Table S2), based on the genome of R. solani, were used to amplify the CYP51 gene fragment of the tebuconazole-sensitive or resistant isolates. I-5TM 2 × High Fidelity Master Mix DNA Polymerase (Molecular Cloning Laboratories, Beijing, China) was used in the PCR. PCR was conducted with a PCR cycle of 98 • C for 3 min, 39 cycles of 98 • C for 10 s, 55 • C for 15 s, and 72 • C for 20 s, ending with an extension at 72 • C for 5 min. PCR products were sequenced (Qingke Biotechnology Co., Ltd., Beijing, China), and the gene sequences of the isolates were measured and analyzed with ClustalX2 software, while the alignment results were visualized with ESPript 3.x software (https://espript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi, accessed on 7 September 2022).

Functional Domain and Structural Analysis of CYP51 Gene
The protein sequences of related fungal CYP51 were downloaded from the NCBI (https: //www.ncbi.nlm.nih.gov/, accessed on 7 September 2022), the conservative functional domains of CYP51 were retrieved by the motif-search (https://www.genome.jp/tools/ motif/, accessed on 7 September 2022) and MEME (https://meme-suite.org/meme/tools/ meme, accessed on 7 September 2022), while the evolutionary tree was constructed with MEGA7.0 software by the maximum likelihood (ML) method [27]. The results were visualized with TBtools software.
Three-dimensional structural modeling of CYP51 from tebuconazole-sensitive isolate was performed by the I-TASSER (http://zhanglab.ccmb.med.umich.edu/I-TASSER, accessed on 7 September 2022), while the three-dimensional structure model of different mutants was built though the Swiss-Model software (https://swissmodel.expasy.org/, accessed on 7 September 2022) with CYP51 model of tebuconazole-sensitive isolate as the template; the tebuconazole structure was downloaded from the PubChem. The binding model and affinity between tebuconazole and the different CYP51 proteins was evaluated using Discovery Studio [28].

Statistical Analysis
The EC50 values for each isolate, cell membrane relative permeability rate, and mycelia growth diameter were compared using analysis of variance (ANOVA) followed by Student's t-test for multiple comparisons (p < 0.05) with the SPSS version 17.0 software package (IBM).

Resistance Frequency of R. solani in Sichuan Province
The results of resistance frequency for all 122 R. solani isolates showed that the resistance frequency to thifluzamide, hexaconazole, tebuconazole, and epoxiconazole was 65.57%, 45.08%, 47.54%, and 36.07%, respectively ( Table 1). The resistance frequency of R. solani in different areas was also different, and the resistance frequency of hexaconazole reached up to 100% in Chengdu Dayi (Table 1). According to the inhibition rate of different isolates to different fungicides, the relationship between tebuconazole and epoxiconazole was higher than that with thifluzamide ( Figure 1).

Resistance Levels of R. solani Isolates to Four Fungicides
The results of resistance levels of four fungicides showed that the EC 50 values of R. solani isolates against thifluzamide were distributed between 0.15 and 0.69 µg/mL, and their resistance ratio (RR) reached 1.43-16.43-fold compared with its sensitive baseline. Most reached sensitive or low resistant levels, apart from MSRS-2-7 with a moderate resistance to thifluzamide isolates (16.43-fold RR); and based on the sensitive baseline of hexaconazole, all isolates showed sensitivity levels with RR values of 2.0-to 4.5-fold, whose EC 50 values ranged from 0.12 to 0.18 µg/mL. According to the sensitive baseline of tebuconazole, whereas many isolates having moderate or high-level resistance were observed (10.59-to 60.78-fold RR), and their EC 50 values ranged from 0.54 and 3.1 µg/mL, in which the highest resistance levels were for MSRS-1-3 and LZHJ-1-8, reaching 52.16-and 60.78-fold, respectively. Furthermore, compared to the sensitivity baseline of epoxiconazole, the RR values of the representative resistant isolates were 1.90-to 35.40-fold, and their EC 50 values were distributed in the range 0.14-3.54 µg/mL. Most retained a sensitive level, in addition to the moderate resistance LZHJ-1-8, which also displayed high level resistance to tebuconazole, of 35.40-fold (Table 3).
At low concentrations of NaCl (0~1.25 g/L), all the mycelia growth diameters of the tebuconazole-sensitive (84-88 mm) and -resistant isolates (57-72 mm) improved with the increase in NaCl concentration; and within the concentration range of 1.25-5 g/L, the mycelia growth diameter of tebuconazole-sensitive isolates (72-85 mm) decreased. This was not consistent with those of tebuconazole-resistant isolates, for which the mycelia growth diameter was increased from 62 to 74 mm. At high concentrations of NaCl that exceeded 5 g/L, all the mycelia growth diameters of tebuconazole-sensitive (8-68 mm) and -resistant isolates (6-58 mm) decreased with the increase in NaCl concentration; until the NaCl concentration was more than 40 g/L, all the isolates could not grow up ( Figure 2B). Although mycelia growth diameters of tebuconazole-resistant isolates were always smaller than those of the tebuconazole-sensitive isolates when the NaCl concentration exceeded 1.25 g/L, the reduction magnitudes of the tebuconazoleresistant isolates (57.83, 66.83, 67.17, and 61.00 mm, respectively) were lower than those of tebuconazole-sensitive isolates (81.00, 79.17, 83.00, and 82.33 mm, respectively), and their difference was extremely significant (p = 0.000 < 0.01). This indicates that the tebuconazole-sensitive isolates were more sensitive to osmotic stress ( Figure 2B).

Functional Domain Analysis of Sterol 14α-Demethylase (CYP51)
The CYP51 of all selected species, except for Coniophora puteana and Stereum hirsutum, contained motif5, motif1, and motif6 domains in series using the Meme-search. Furthermore, there was no other domain between motif5 and motif1, which were composed of 50 amino acid residues; motif6 was composed of 36 amino acid residues and possessed absolutely conserved amino acid residues (EXLR, a helix K motif); and the heme-binding signature motif (PFxxGxxxCxG) was located in motif3 which was behind motif6 (Figures 4 and S1). According to the evolutionary tree and gene structure diagram, R. solani CYP51 had high homology with Heliocybe sulcate, Gloeophyllum trabeum, Fomitiporia mediterranea, and Sanghuangporus baumii, showing a similar gene structure ( Figure  4).

Functional Domain Analysis of Sterol 14α-Demethylase (CYP51)
The CYP51 of all selected species, except for Coniophora puteana and Stereum hirsutum, contained motif5, motif1, and motif6 domains in series using the Meme-search. Furthermore, there was no other domain between motif5 and motif1, which were composed of 50 amino acid residues; motif6 was composed of 36 amino acid residues and possessed absolutely conserved amino acid residues (EXLR, a helix K motif); and the heme-binding signature motif (PFxxGxxxCxG) was located in motif3 which was behind motif6 (Figures 4 and S1). According to the evolutionary tree and gene structure diagram, R. solani CYP51 had high homology with Heliocybe sulcate, Gloeophyllum trabeum, Fomitiporia mediterranea, and Sanghuangporus baumii, showing a similar gene structure (Figure 4).

Discussion
Fungicide applications have been commonly used for the control of R. solani in China. However, the irrational and frequent usage of fungicide has caused a more serious problem: the proliferation of resistance genes [29]. There have been many reports about the resistance to different types of fungicides in R. solani, such as QoI fungicides [4]; however, little is known about the resistance to SDHI and DMI fungicides [9]. Our results showed that the EC50 values of the sensitive isolates were 0.03-0.05 μg/mL, consistent with the findings of Chen et al. (2012) [7], and the resistance levels of most resistant isolates were less than 5-fold, in addition to MSRS-2-7. At the same time, we also found that almost all the screened tebuconazole-resistant isolates reached the moderate level of resistance, especially LZHJ-1-8 isolate, which was not only highly resistant to

Discussion
Fungicide applications have been commonly used for the control of R. solani in China. However, the irrational and frequent usage of fungicide has caused a more serious problem: the proliferation of resistance genes [29]. There have been many reports about the resistance to different types of fungicides in R. solani, such as QoI fungicides [4]; however, little is known about the resistance to SDHI and DMI fungicides [9]. Our results showed that the EC50 values of the sensitive isolates were 0.03-0.05 µg/mL, consistent with the findings of Chen et al. (2012) [7], and the resistance levels of most resistant isolates were less than 5-fold, in addition to MSRS-2-7. At the same time, we also found that almost all the screened tebuconazole-resistant isolates reached the moderate level of resistance, especially LZHJ-1-8 isolate, which was not only highly resistant to tebuconazole, but also highly resistant to epoxiconazole. Resistance to DMI fungicide in many plant pathogens is of a quantitative nature characterized by slow shifts in sensitivity toward resistance [30].
Tebuconazole and epoxiconazole belong to DMI fungicides, which have been proven to bind with the heme part of CYP51 and inhibit demethylation of 24-methylenedihydrolanosterol, a precursor of the cell membrane component ergosterol [31,32]. These are able to disrupt the cell membranes, causing an increase in relative electric conductivity [33]. Our results found that the relative electric conductivity of both tebuconazole-resistant and -sensitive isolates showed a significant increase tendency after tebuconazole treatment; meanwhile, we also found that although the tebuconazole-resistant isolates had an increased ability to tolerate tebuconazole and high osmotic pressure, their growth rate and relative electric conductivity with low sugar or tebuconazole were inferior to those of the sensitive strains, showing a fitness cost. Shao et al. (2015) [34] found that the laboratoryinduced fluazinam-resistant mutants of B. cinerea were more sensitive to the osmotic stress than their fluazinam-sensitive parental isolates. Karaoglanidis et al. (2011) [35] found that the resistance to tebuconazole isolates had a significant adverse effect on the mycelial growth rate and pathogenicity.
We speculated that the resistance to DMI fungicides and its fitness cost is due to the pleiotropy caused by the mutation of the target CYP51 [36]. Some CYP51 mutations reduce the affinity between the target protein and pesticide, which is one of the important factors leading to the production of resistance [37], and may also affect fungal sterol synthesis and, thus, its fitness [38]. Our CYP51 mutation detection results showed that all the screened tebuconazole-resistant isolates had site mutations, while CYP51 S94A,N406S,H793R mutations were the main resistant population types. Pereira et al. [39] found that the resistance to tebuconazole had a significant linear correlation with the G461S mutation frequency. Stammler et.al (2012) [40] indicated that some mutations in CYP51 (e.g., mutations A379G, I381V), were known to be an adaptive response to DMIs that had already disappeared in Northern Europe. Other mutations in the CYP51 gene, e.g., G143A in V136A, A379G, I381V, and mutations or deletions at the amino acid positions 459-462 of CYP51 in M. graminicola [41], Y134F in Puccinia triticina [42], Z. tritici [43], Y137H in Fusarium graminearum [44] etc., were proven to be related with DMI fungicide resistance. The K motif and heme-binding motif constituted the conserved domains characteristic of P450 proteins [45]. Although S94A, N406S, L750P, and H793R were not located in these motifs, six substrate recognition sites (SRS1-6) have been identified to contain the amino acid residue S, N, or L [46]. Our results also showed that the binding energy of CYP51 and tebuconazole in the sensitive isolate was significantly less than that of CYP51 S94A,N406S,H793R and CYP51 S94A,N406S,L750P,H793R . Therefore, the point mutation on the target gene CYP51 of tebuconazole was associated with the resistance of rice bacterial strain to tebuconazole. In the next step, we will use site-directed mutagenesis to edit CYP51 of R. solani to verify the relationship between these mutations and resistance, and to explore the mechanism by which mutations lead to changes in the fitness cost and resistance.

Conclusions
The resistance frequency results of 122 R. solani isolates by MIC showed that the resistance frequency for several fungicides was different, and the resistance frequency of tebuconazole reached 47.54%. Furthermore, a moderate resistance to thifluzamide was identified in isolate MSRS-2-7, whereas most isolates were sensitive to hexaconazole or epoxiconazole. In addition, a highly tebuconazole-resistant isolate LZHJ-1-8 displayed a moderate resistance to epoxiconazole, and the screened tebuconazole-resistant isolates showed a moderate or high resistance to tebuconazole. The tebuconazole-resistant isolates showed higher fitness costs with a lower growth rate and higher relative electric conductivity, demonstrating an increased ability to tolerate tebuconazole and high osmotic pressure. CYP51 mutation results showed that the tebuconazole-resistant isolates retained S94A, N406S, and H793R mutations, while LZHJ-1-8 possessed another mutation, L750P, which supported the lowest binding energy with tebuconazole. These results suggest that S94A, N406S, L750P, and H793R mutations of CYP51 may be linked to the resistance to tebuconazole.