Wheat sheath eyespot (WES) is an important disease in wheat production. Due to the lack of wheat varieties with stable resistance and high yield, chemical agents are currently the main control method. Trifloxystrobin has good control effects on a variety of plant pathogenic fungi due to its wide fungicidal spectrum and high fungicidal activity. Laboratory preliminary studies have shown that trifloxystrobin has good antibacterial activity against Rhizoctonia cerealis, but is there a risk of drug resistance after long-term application? And what is the mechanism by which R. cerealis develops resistance to it? Both need further study. In this study, the trifloxystrobin-resistant R. cerealis obtained by drug domestication in the early stage of the laboratory was used as the main research material to study the biological characteristics and R. cerealis resistance mechanism to trifloxystrobin. Our results showed that compared with the sensitive isolates, the mycelial growth rate of the trifloxystrobin-resistant R. cerealis mutant was reduced, the ability to produce sclerotia was enhanced, the pathogenicity to the host wheat was enhanced, and it was more sensitive to temperature. At the same time, the results of osmotic pressure measurement showed that compared with the sensitive strain, the mycelial growth rate of the trifloxystrobin-resistant R. cerealis mutant under different pH and salt stresses had no significant difference, while low concentration (< 20 g/L) of glucose stress promoted the mycelial growth of the trifloxystrobin-resistant R. cerealis mutants to a certain extent. In addition, there was no cross-resistance between trifloxystrobin-resistant R. cerealis and the commonly used fungicides difenoconazole, flufenoxazole, flutriafol, tebuconazole, metconazole and carbendazim, and there was negative cross-resistance between it and propiconazole, hexaconazole, thiofuran and fluazinam, that is, the above fungicides can be used as alternative fungicides in areas where wheat sheath blight is resistant to trifloxystrobin. The research results provide data support for clarifying the resistance mechanism of R. cerealis to trifloxystrobin, which is of great significance for effectively preventing and controlling wheat sheath blight and ensuring the safe production of wheat.
Article
Exploring the mechanism of resistance of Rhizoctonia cerealis to trifloxystrobin
https://doi.org/10.21203/rs.3.rs-4721628/v1
This work is licensed under a CC BY 4.0 License
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Wheat sheath eyespot
Rhizoctonia cerealis
Trifloxystrobin
Biological characteristics
Cross-resistance
Wheat (Triticum aestivum L.) is one of the most important food crops in the world. It occupies an important position in global food crops because it is rich in starch, protein, dietary fiber and multiple vitamins [1]. According to the FAO report, the total world wheat production in 2022 is 783.9 million tons, and it emphasizes that the safe production of wheat and other food crops plays an important role in ensuring world food security [2]. At the same time, wheat is also one of the three major food crops in my country. According to incomplete statistics, the total wheat production in my country in 2022 alone is about 138 million tons, accounting for about 18% of the world's total wheat production [3]. At present, due to the influence of comprehensive factors such as global warming and adjustment of planting structure, soil-borne plant diseases represented by wheat sheath blight and stem base rot are seriously threatening the safe production of wheat [4]. Among them, wheat sheath blight caused by Rhizoctonia cerealis and Rhizoctonia solani as dominant fungal sources is an important fungal soil-borne disease in current wheat production. It has the characteristics of high incidence, wide spread and great harm, which seriously threatens the safe production of wheat [4]. Studies have shown that wheat sheath blight occurs in all major wheat-producing areas in my country, with a general incidence of 10%-20%, but the incidence is as high as 70% when the air humidity is high and nitrogen fertilizer is excessively applied [5, 6]. At present, because the newly bred wheat varieties cannot completely resist wheat sheath blight [7], the current prevention and control of wheat sheath blight in production is still mainly chemical control [8]. Studies have shown that fungicides such as tebuconazole, diniconazole, propiconazole, jinggangmycin, and thiofuran have good control effects on wheat sheath blight [9, 10, 11, 12]. However, if chemical fungicides are applied for a long time, in large quantities, and unscientifically, the pathogens will develop resistance to the fungicides, resulting in reduced control effects or even failure of control.
Trifloxystrobin is a new type of fluorine-containing fungicide developed by Syngenta in 1998 [13]. It has the characteristics of high efficiency, broad spectrum, protection, treatment, eradication, penetration, systemic activity, resistance to rainwater erosion, and long lasting effect, and has good control effects on many pathogenic fungi [14]. Studies have shown that trifloxystrobin has a good control effect on strains of plant pathogenic fungi that are resistant to 1,4-demethylase inhibitors, benzamides, dicarboxylamines and benzimidazoles [15]. According to incomplete statistics, trifloxystrobin has a good control effect on a variety of plant pathogenic fungi including wheat powdery mildew, rust, glume blight and rice blast [13]. Preliminary laboratory studies have shown that R. cerealis has good antibacterial activity against trifloxystrobin, with an average EC50 value of about 4.09 µg/mL for more than 80 test strains, indicating that trifloxystrobin has the potential to prevent and control wheat sheath blight. However, whether long-term application of trifloxystrobin will cause the risk of R. cerealis developing resistance to it? And what is the mechanism by which R. cerealis develops resistance to it? These are not yet fully understood and need further study.
This study intends to use more than 80 strains of R. cerealis isolated from wheat sheath blight diseased plants in different cities in Henan Province collected in the laboratory in the early stage, as well as mutant strains of R. cerealis with high level resistance to trifloxystrobin obtained in the early stage of the laboratory through fungicide domestication as the main research materials, to study the resistance mechanism of R. cerealis to trifloxystrobin, in order to clarify the biological characteristics of R. cerealis resistant to trifloxystrobin, and further explore the mechanism of resistance of R. cerealis to trifloxystrobin, which is of great significance for the effective prevention and control of Wheat sheath eyespot and ensuring the safe production of wheat.
2.1 Test fungicides
The fungicides tested were mainly 95.0% fenpropimorph technical (purchased from Hubei Jianyuan Chemical Co., Ltd.), 95.0% propiconazole technical (provided by Guangxi Tianyuan Biochemical Co., Ltd.), 95.9% fluazifop technical (provided by Guangxi Tianyuan Biochemical Co., Ltd.), 98.0% fluopicolide technical (provided by Guangxi Tianyuan Biochemical Co., Ltd.), 95.0% hexaconazole technical (provided by Guangxi Tianyuan Biochemical Co., Ltd.), 95.5% thiofuran technical (provided by Guangxi Tianyuan Biochemical Co., Ltd.), 96.0% tebuconazole technical (purchased from Jiangsu Sheyang Huanghai Pesticide Chemical Co., Ltd.), 96.0% metconazole technical (purchased from Hubei Maoerwo Biomedicine Co., Ltd.) and 98.0% fluazifop technical (provided by Guangxi Tianyuan Biochemical Co., Ltd.). All of the above technicals were dissolved in acetone (analytical grade, purchased from Tianjin Fuyu Fine Chemical Co., Ltd.). In addition, 98.1% carbendazim technical (purchased from Hubei Maoerwo Biopharmaceutical Co., Ltd.) was tested, which was mainly dissolved in 0.1 mol/L hydrochloric acid (analytical grade, purchased from Tianjin Fuyu Fine Chemical Co., Ltd.).
2.2 Test strains and culture medium
The sensitive strains of R. cerealis to trifloxystrobin, XMWK2023YJ-33, XMWK2023YJ-45, XMWK2023YJ-68, and XMWK2023YJ-75, with EC50 values of 0.292, 0.863, 0.201, and 0.308 µg/mL, were collected from wheat plants with typical sheath blight symptoms in the main wheat producing areas of Henan Province in 2023 and analyzed. The EC50 values of the tested R. cerealis mutants SDQO-18-1-1, SDQO-18-1-2, SDQO-18-1-3, and SDQO-18-1-4 resistant to trifloxystrobin were 914.729, 1779, 879.21, and 384.497 µg/mL, respectively, which were obtained in the laboratory through pharmaceutical domestication and have been preserved in the laboratory since 2023.
The preparation method of potato dextrose agar (PDA) medium is as follows: weigh 200 g of fresh peeled potatoes, cut into small pieces and put into 1 L of water and boil for 20–30 min, then filter with gauze, add 18.0 g of agar and 20.0 g of glucose to the filtrate, stir until completely melted, add water to make up to 1 L, divide into conical bottles, seal with sealing film, sterilize in a pressure cooker (fully automatic high-pressure steam sterilizer (HVA-85), HIRAYAMA, Japan) at 121℃ and 101.3 Kpa for 20 min, then cool and set aside.
2.3 Biological characteristics research
The biological characteristics of the sensitive strains of R. cerealis (XMWK2023YJ-33, XMWK2023YJ-45, XMWK2023YJ-68 and XMWK2023YJ-75) and the trifloxystrobin-resistant R. cerealis mutants (SDQO-18-1-1, SDQO-18-1-2, SDQO-18-1-3, and SDQO-18-1-4) were compared by measuring the mycelial growth rate on PDA plates, the ability to produce sclerotia on PDA culture medium and the pathogenicity to the base of wheat stems.
2.3.1 Mycelial growth rate assay
This study adopted the method reported by Chen Yi et al. [16] with slight modifications. The main steps are as follows: After the test strain is cultured on PDA for 3–5 days, a hole is punched using a hole puncher, and a 7 mm diameter mycelium block is inoculated on the PDA plate. After culturing at 25°C and in the dark for 24 h, 48 h and 72 h, the colony diameter is measured using the cross method and the average value is taken.
2.3.2 Sclerotium production ability assay
This study adopted the method reported by Chen Yi et al. [16] with slight modifications. The main steps are as follows: After the test strain is cultured on the PDA plate for 3–5 days, a hole is punched using a hole puncher, and a mycelium block with a diameter of 7 mm is inoculated on the PDA plate, and cultured at 25°C in the dark. After 5 days, the sclerotia production is observed and recorded every day.
2.3.3 Determination of the pathogenicity of R. cerealis resistant and susceptible to trifloxystrobin to host wheat
This study was conducted using the method reported by Chen Yi et al. [16] with slight modifications. The main steps are as follows: Take an appropriate amount of wheat grains, soak them at room temperature for 3 days, then rinse them with water 3 times, weigh the same weight of wheat grains and place them in a 250 mL triangular flask, sterilize them at high temperature (121℃, 45 min), and then inoculate 3 ~ 5 pieces of cakes of sensitive strains of R. cerealis to trifloxystrobin and mycelial blocks of resistant mutants cultured on PDA medium for 4 ~ 5 days, inoculate 2 bottles with each strain, culture at 25 ℃ for 14 days, and shake the triangular flask every day to prevent wheat grain agglomeration. Sow seeds (Bai Nong 207) in a 15 cm diameter plastic pot filled with sterilized soil, 20 seeds per pot, cover with sterilized soil, cultivate and manage in a greenhouse, and inoculate pathogens when wheat is in the second leaf stage. Each test is repeated 3 times, and the disease is investigated 30 days after inoculation. (Note: We have permission to collect Wheat Plant.)
2.4 Osmotic sensitivity assay
This study used the method reported by Chen Yi et al. [16] to determine the effects of different concentrations of salt, sugar, temperature and pH on mycelial growth, with slight modifications. The specific method is as follows.
2.4.1 Effects of high salt (NaCl) medium on mycelial growth
The test strains were inoculated on PDA plates containing different salt concentrations (5 g/L, 10 g/L, 20 g/L and 40 g/L) and cultured at 25°C in the dark for 3 to 5 days. The colony diameter was measured by the cross method. The plate without NaCl was used as the control. Each treatment was repeated 3 times.
2.4.2 Effects of high sugar (glucose) medium on mycelial growth
The test strains were inoculated on PDA plates containing different glucose concentrations (10, 20, 40, 80, 100, and 150 g/L), cultured at 25°C in the dark for 3–5 days, and the colony diameter was measured by the cross method, with no glucose as the control. Each treatment was repeated 3 times.
2.4.3 Effects of different temperatures on mycelial growth
The test strains were inoculated on PDA plates and cultured at 5°C, 15°C, 25°C and 37°C for 3–5 days. The colony diameters were measured using the cross method, with 3 replicates for each treatment.
2.4.4 Effect of pH on mycelial growth
The test strains were inoculated on PDA plates with pH values of 4, 7 and 11, respectively, and cultured at 25°C in the dark for 3–5 days. The colony diameters were measured using the cross method, with three replicates for each treatment.
2.5 Cross-resistance assay
This study was conducted using the Spearman rank correlation coefficient method reported by Wang Kaijun et al. [27], with slight modifications. The main steps are as follows: the test strain was inoculated on a PDA plate and cultured at 25°C in the dark for 3–5 days. A mycelium block with a diameter of 7 mm was taken from the edge of the colony using a hole puncher and inoculated on a PDA plate containing different concentrations of the trifloxystrobin. The plate without the drug was used as the control group and cultured at 25°C in the dark for 3–5 days. The colony diameter was then measured using the cross method and the average value was taken. Three replicates were set for each treatment. The settings of different drug concentrations are shown in (Table 1). The result judgment criteria are as follows: when the sensitivity level value P < 0.05, the result is credible; ρ > 0.8, there is obvious cross-resistance between the two fungicides; 0.5 ≤ ρ ≤ 0.8, there is moderate correlation between the two agents; 0.3 ≤ ρ ≤ 0.5, there is low correlation between the two agents; 0 < ρ < 0.3, the correlation between the two agents is poor; ρ < 0, there is negative cross-resistance between the two fungicides.
2.6 Statistical analysis
Experimental data were conducted using SPSS software (17.0), Fisher's least significant difference test was used to analyze significant differences (p ≤ 0.05), and GraphPad.Prism (9.0) software was used for drawing.
3.1 Analysis on the biological characteristics of R. cerealis resistant to trifloxystrobin
3.1.1 Mycelial growth rate of R. cerealis resistant to trifloxystrobin
The colony morphology of the test strains after culturing on PDA plates for 3 to 5 days is shown in Fig. 1. On the first day after each test strain was inoculated on the PDA plate, there was no significant difference in the mycelium growth rate of each strain, which was about 1.35 cm. 2 days after inoculation, there was a significant difference in mycelial growth rate between the trifloxystrobin-sensitive strain of Rhizoctonia cerealis and the trifloxystrobin-resistant R. cerealis mutant strain. The silk growth rates were lower than those of R. cerealis susceptible strains to trifloxystrobin. There are certain differences in mycelial growth rates among the strains of R. cerealis that are sensitive to trifloxystrobin. The growth rates of R. cerealis strains sensitive to trifloxystrobin XMWK2023YJ-68 and XMWK2023YJ-75 are fast. Among XMWK2023YJ-33 and XMWK2023YJ-45, strains of R. cerealis that are sensitive to trifloxystrobin, XMWK2023YJ-68, the strain that is sensitive to trifloxystrobin of R. cerealis, has the fastest mycelium growth rate, while XMWK2023YJ-45 has the fastest hyphae growth rate. Mycelium grows the slowest. In addition, compared with the trifloxystrobin-resistant R. cerealis mutant strain, the trifloxystrobin-resistant R. cerealis mutant strain has sparse hyphal growth and hyphal growth. The amount is less.
3.1.2 Analysis on the Sclerotium-producing capacity of R. cerealis resistant to trifloxystrobin
By analyzing the sclerotia-producing ability of the tested strains, the results showed that the sclerotia production time of the trifloxystrobin-resistant R. cerealis mutants was earlier than that of the tested wild-type strain, and the number of sclerotia produced was significantly higher. Compared with the wild-type strain, there are also certain differences in the time and number of sclerotia produced between different strains. Among them, the trifloxystrobin-resistant R. cerealis mutants SDQO-18-1-1 and SDQO-18-1-3 began to produce sclerotia on the 6th day after the strain was cultured (the sclerotia were white at first, and as the culture time (the sclerotia color gradually changes to dark brown), the sclerotia production time of mutant SDQO-18-1-2 is slightly later, and sclerotia begins to be produced after 7 days of culture, but the mutant SDQO-18-1-4 strain The production time of sclerotia on PDA was the latest, and sclerotia were only found sporadically in the PDA culture medium after 8 days of culture. The number of sclerotia produced at the beginning was significantly less than that of SDQO-18-1-1 and SDQO-18- 1 -2 and SDQO-18-1-3 (Fig. 2).
3.1.3 Analysis of the pathogenicity of R. cerealis resistant to trifloxystrobin
The test results of the pathogenicity of R. cerealis strains to the host wheat show that there is a significant difference in the pathogenicity of the R. cerealis mutant strains to the trifloxystrobin-sensitive strains of R. cerealis to the host wheat. The pathogenicity of the trifloxystrobin-resistant mutant R. cerealis strain to the host wheat was significantly stronger than the trifloxystrobin-sensitive strain of R. cerealis. Among them, there are certain differences in the pathogenicity of different strains of R. cerealis to trifloxystrobin on the host wheat. The pathogenicity of XMWK2023YJ-75 strain of R. cerealis to trifloxystrobin on the host wheat The strongest potency, significantly stronger than the three strains of R. cerealis that are sensitive to trifloxystrobin, namely XMWK2023YJ-68, XMWK2023YJ-45 and XMWK2023YJ-33. However, the four trifloxystrobin-resistant mutants, SDQO-18-1-1, SDQO-18-1-2, SDQO-18-1-3 and SDQO-18-1-4, had no significant pathogenicity to the host wheat. Differences in penetration (Fig. 3).
3.2 Analysis of the osmotic pressure sensitivity of R. cerealis resistant to trifloxystrobin
3.2.1 Effects of high salt (NaCl) medium on mycelial growth of test isolates
By carrying out mycelial growth determination of trifloxystrobin-resistant R. cerealis under a series of salt stress conditions, the results showed (Fig. 4): compared with the sensitive strain, the resistant mutant was tested at 5, 10, 20, 40, Under the salt stress concentration of 60 g/L, it did not show a tendency to be more tolerant to high salt stress, but there were certain differences among different strains. Compared with the control group, under the salt stress concentration of 5 g/L, the mycelium growth of both the sensitive strain and the resistant mutant strain was inhibited, and the colony morphology was not significantly different from that of the control group. Among them, the inhibition of the XMWK2023YJ-45 strain, the inhibition rate is about 38%; the inhibition rate against XMWK2023YJ-75 strain is about 16%; the inhibition rate against SDQO-18-1-3 strain is about 41%; while the inhibition rate against XMWK2023YJ-33, XMWK2023YJ-68, SDQO-18- The inhibition rate of 1–1, SDQO-18-1-2 and SDQO-18-1-4 strains is about 20%. When the salt stress concentration reaches 40 g/L, the creeping hyphae of the sensitive strain and mutant strain stop growing, and only a small amount of mycelium grows upward. When the salt stress concentration reached 60 g/L, mycelial growth almost completely stopped, and the inhibition rate was as high as 93%.
3.2.2 Resistant and susceptible to trifloxystrobin Effects of high sugar (glucose) medium on mycelial growth of test isolates
The test of mycelial growth of R. cerealis resistant and sensitive to trifloxystrobin on PDA culture medium with different glucose content showed that the mycelial growth of the tested R. cerealis strains sensitive to trifloxystrobin and the mutant R. cerealis strains resistant to trifloxystrobin were more sensitive to high-sugar PDA medium(Fig. 5). Compared with the mutant strains of R. cerealis resistant to trifloxystrobin, the strains of R. cerealis sensitive to trifloxystrobin were more sensitive to high-sugar environment. As shown in Fig. 5, when the concentration of glucose in the PDA medium is between 0 and 20 g/L, as the sugar concentration in the PDA medium increases, the mycelial growth of the strain of R. cerealis that is sensitive to trifloxystrobin is first inhibited and then promoted, while the mycelial growth of the mutant strain of R. cerealis that is resistant to trifloxystrobin is first promoted and then inhibited; when the concentration of glucose in the PDA medium is ≥ 20 g/L, as the sugar concentration in the PDA medium increases, the mycelial growth of the strain of R. cerealis that is sensitive to trifloxystrobin and the mutant strain of R. cerealis that is resistant to trifloxystrobin is inhibited. When the glucose concentration in the PDA culture medium was 80 g/L, the inhibition rate of mycelial growth of four strains of R. cerealis, XMWK2023YJ-33, XMWK2023YJ-45, XMWK2023YJ-68, and XMWK2023YJ-75, which were sensitive to trifloxystrobin, was about 35%, while the inhibition rate of mycelial growth of mutants SDQO-18-1-1 and SDQO-18-1-2 was about 75%, the inhibition rate of mutant SDQO-18-1-3 was about 57%, and the inhibition rate of mutant SDQO-18-1-4 was about 42%.
3.2.3 Resistant and susceptible to trifloxystrobin Effects of different temperatures on mycelial growth of tested isolates
The mycelial growth test of R. cerealis strains resistant to and susceptible to trifloxystrobin at different temperatures showed that: the sensitivity of R. cerealis to trifloxystrobin and the resistance to trifloxystrobin of R. cerealis at low and high temperatures were There was a significant inhibitory effect on mycelial growth, and the optimal growth temperature of the trifloxystrobin-resistant mutant strain of R. cerealis and the trifloxystrobin-sensitive strain of R. cerealis was both at 25°C (Fig. 6), the trifloxystrobin-sensitive strain of R. cerealis is more sensitive to temperature changes than the trifloxystrobin-resistant R. cerealis mutant.
3.2.4 Effect of pH on the mycelial growth of tested isolates
The experimental determination of hyphae growth of R. cerealis isolates resistant to and susceptible to trifloxystrobin at different pH values showed (Fig. 7): except for the trifloxystrobin-resistant R. cerealis mutant strain SDQO-18-1-3, the alkaline environment had an inhibitory effect on the hyphae growth of R. cerealis strains sensitive to trifloxystrobin and R. cerealis mutant strains resistant to trifloxystrobin. The acidic environment had a significant promoting effect on the hyphae growth of R. cerealis strains XMWK2023YJ-33 and XMWK2023YJ-68, which were sensitive to trifloxystrobin, and had a certain inhibitory effect on the hyphae growth of R. cerealis strain XMWK2023YJ-75, which was sensitive to trifloxystrobin. It had a certain inhibitory effect on SDQO-18-1-1 and SDQO-18-1-2, and promoted the hyphae growth of SDQO-18-1-3 and SDQO-18-1-4. In an alkaline environment, the mycelial growth of R. cerealis XMWK2023YJ-33, XMWK2023YJ-45, XMWK2023YJ-68 and XMWK2023YJ-75 and the oxathiapiprolin-resistant mutant strains SDQO-18-1-1, SDQO-18-1-2 and SDQO-18-1-4 were inhibited, but the mycelial growth of the oxathiapiprolin-resistant mutant SDQO-18-1-3 was promoted to a certain extent.
3.3 Analysis of cross-resistance between R. cerealis mutants resistant to trifloxystrobin and other commonly used fungicides
The study showed that trifloxystrobin had strong antibacterial activity against trifloxystrobin-sensitive strains of R. cerealis XMWK2023YJ-33, XMWK2023YJ-45, XMWK2023YJ-68 and XMWK2023YJ-75, with EC50 values of 0.292, 0.863, 0.201 and 0.308 µg/mL, respectively. However, its performance in trifloxystrobin-resistant R. cerealis mutant strains was not as good as that in the strains of R. cerealis sensitive to trifloxystrobin, and its efficacy was greatly reduced, with EC50 values of 914.729, 1779, 879.21 and 384.497 µg/mL, respectively. According to the Spearman rank correlation method analysis, when the level value P > 0.05, there is no cross-resistance between the two fungicides, and the P value of trifloxystrobin and difenoconazole is 0.7660, that is, trifloxystrobin and difenoconazole There is no cross-resistance between difenoconazole and difenoconazole. The P values of triflstrostrobin and epoxiconazole, triflustroconazole, tebuconazole, tebuconazole and carbendazim were 0.5975, 0.0754, 0.4082, 0.0741 and 0.0827 respectively, with all P values > 0.05, that is, trifloxystrobin and there is no cross-resistance between epoxiconazole, triconazole, tebuconazole, myconazole and carbendazim. The P values of trifloxystrobin for propiconazole and hexaconazole are 0.0048 and 0.0030, respectively, and the P values for thifuramide and fluazinam are < 0.0001; the P values are all less than 0.05, and its ρ values are − 0.8719 and − 0.8911 respectively. -0.9767 and − 0.9731, and the ρ values are all < 0, indicating that there is negative cross-resistance between trifloxystrobin and propiconazole, hexaconazole, thifuramide and fluazinam. Although the trifloxystrobin-resistant R. cerealis mutants SDQO-18-1-1, SDQO-18-1-2, SDQO-18-1-3, and SDQO-18-1-4 strains are susceptible to trifloxystrobin. There have been significant changes in resistance, but no evidence of cross-resistance between trifloxystrobin and other fungicides has been found so far (Fig. 8).
Wheat (Triticum aestivum L.) is one of the three major food crops in the world. About 35%-40% of the world's population relies on wheat as their main food [17]. China as a largest wheat planting area in the world, with a maximum wheat planting area of 30 million hectares, accounting for 13.5% of the world's total wheat planting area [31],which is also the country with the largest wheat production in the world, accounting for about 18% of the world's total wheat production. In 2022 alone, my country's total wheat production reached 138 million tons [17]. Meanwhile, China is also the world's largest wheat consumer, with an average annual consumption of 122 million tons of wheat. Therefore, the safe production of wheat is directly related to China's food security and social stability [28].
However, wheat growth and development is accompanied by a variety of diseases and pests, which restrict the high quality and high yield of wheat. Wheat sheath eyespot is one of the serious soil-borne diseases. Wheat sheath eyespot is a fungal disease caused by R. cerealis and R. solani infecting wheat and is widely distributed in the world. In recent years, under the influence of comprehensive factors such as global warming and adjustment of planting structure, fungal diseases mainly caused by wheat sheath blight have had a great impact on the safe production of wheat [18]. Studies have shown that wheat sheath blight occurs in major wheat producing areas in my country, with a general incidence rate of 10%-20%, and in the case of high air humidity and excessive application of nitrogen fertilizer, the incidence rate is even as high as 70% [5]. At present, because the newly bred wheat varieties cannot completely resist wheat sheath blight [7], the current prevention and control of wheat sheath blight in production is still mainly chemical control [8].
Trifloxystrobin is a new type of fluorine-containing fungicide developed by Syngenta in 1998. It belongs to the methoxyacrylate fungicide class [13]. It inhibits mitochondrial electron transfer by binding to the cytb and c1 complex Qo sites of the mitochondrial species of pathogenic cells, thereby hindering ATP production and acting on pathogens [19, 20]. Strobilurin fungicides exert their antibacterial effect by inhibiting mitochondrial respiration, so they have a strong inhibitory effect on most fungi and are widely used in the prevention and control of various diseases of cereals, vegetables, and fruit trees [21, 22]. Dong Liying [23] et al. found that the use of 28% tricyclazole·pyraclostrobin SC has a good preventive and therapeutic effect on rice seedling blast and panicle blast, and the highest prevention effect on panicle blast in the field reached 91.11%; Clough [24] and other previous studies showed that 250 g/L azoxystrobin suspension, 25% oxathiobol-butyl emulsifiable concentrate and 250 g/L pyraclostrobin emulsifiable concentrate 1000 times the foliage spray 7 days after the prevention of grape downy mildew was above 90.17%, which has good prevention effect; Jiang [25] and other studies have shown that pyraclostrobin has a strong inhibitory effect on the growth of sesame Fusarium hyphae. Preliminary laboratory studies have shown that trifloxystrobin has good antibacterial activity against R. cerealis. Its EC50 values for more than 80 strains ranged from 0.03 to 19.57 µg/mL, with an average EC50 value of 4.09 µg/mL, indicating that trifloxystrobin-methyl has the potential to prevent and control wheat sheath eyespot.
This study studied the fitness differences between trifloxystrobin-resistant mutant strains of R. cerealis and trifloxystrobin-sensitive strains of R. cerealis in terms of adaptability to temperature, high sugar, high salt, and pH. It was found that the hyphae of the trifloxystrobin-resistant mutant strain of R. cerealis are sparser and the amount of mycelium is less than that of the trifloxystrobin-sensitive strain of R. cerealis, which is consistent with Jiang [25], and the same findings have been reported by et al. In addition, the adaptability of the trifloxystrobin-resistant mutant strain to temperature is significantly higher than that of the trifloxystrobin-sensitive strain of R. cerealis. In terms of adaptability to different pH environments, high salt, high sugar and other stresses, The trifloxystrobin-resistant mutant strain of R. cerealis is also slightly stronger than the trifloxystrobin-sensitive strain of R. cerealis, which is the same as the results of previous studies [7, 16]. During the research process, it was also found that the resistant strains also had significant differences in the presence or absence of aerial mycelium and the ability to produce sclerotia. That is, compared with the susceptible strains of R. cerealis to trifloxystrobin, the resistant strains were The hyphal growth of the mutant strain of R. cerealis is sparse and the amount of hyphae is less; while the degree of colony deformity and sclerotia production capacity of the trifloxystrobin-resistant R. cerealis mutant strain are also significantly higher than those of the parent susceptible strain.
As a member of the strobilurin fungicide class, trifloxystrobin has the advantages of a broad fungicide spectrum, a single site of action, high fungicide activity, and both protective and therapeutic effects. It is also a ‘high resistance risk fungicide’, but there are no reports of cross-resistance between trifloxystrobin and other commonly used fungicides. Studies have shown that the combination of strobilurin fungicides and triazole fungicides can improve the control effect of wheat fusarium head blight [26]. Therefore, it can be used in combination with other triazole fungicides to reduce or delay the development of resistance to trifloxystrobin in the test pathogens.
There are many factors that affect biological fitness. This study only selected some of the influencing factors for study, and did not further study the molecular mechanism of its resistance and the gene expression level of the target gene CYTB in the strains resistant to trifloxystrobin [29, 30]. In subsequent studies, the CYTB gene, the site of action of methoxyacrylate fungicides on R. cerealis, can be studied to further improve the research on the risk of resistance of R. cerealis to trifloxystrobin.
Author Contributions: Conceptualization, Y.-C. Shi., F. Zhou. and R.-Q. Liu.; methodology, F. Zhou. and S.-C. Xu.; software, S.-C. Xu. and C.-W. Li.; formal analysis, S.-C. Xu. and M.-H. Sun.; investigation, F. Zhou., J.-L. Zhang. and S.-C. Xu.; resources, F. Zhou.; data curation, S.-C. Xu., Z.-K. Li. and T.-T. Qiao.; writing-original draft preparation, F. Zhou.; writing-review and editing, C.-W. Li., R.-Q. Liu., S.-C. Xu. and F. Zhou.; visualization, S.-C. Xu.; supervision, F. Zhou., R-Q. Liu. and C.-W. Li.; project administration, F. Zhou. and R.-Q.Liu.; funding acquisition, F. Zhou. and R.-Q. Liu. All authors have read and agreed to the published version of the manuscript.
Funding: This work was sponsored by the Henan Province University Innovation Talents Support Program (No. 24HASTIT059), the special fund project for central guiding Henan province local development (No. 20221343034), the Henan provincial science and technology major project (No. 221100110100), and the postdoctoral research project in Henan province (No. HN2022109).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Conflicts of Interest: The authors declare no conflict of interest.
Statement: The authors confirm that the data supporting the findings of this study are available within the article [and/or its supplementary materials].
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No competing interests reported.