Seeds of the glutinous sorghum variety Qiangao 8 were obtained from the Guizhou Subcenter of Chinese Wheat Improvement. The seeds were washed with tap water to remove any fungicidal residues from the surface of the seeds and then dried at room temperature. The dried seeds were then surface sterilized with 1% sodium hypochlorite in 10% ethanol for 10 min, rinsed with sterile distilled water three times, and immersed in sterile distilled water for 4 h. This was followed by a hot water treatment in sterile distilled water at 60°C for 5 min to kill any endophytic fungi inside the seeds. Lastly, the seeds were rinsed with sterile distilled water, air-dried, and stored at 4°C until use (Huang and Backhouse, 2005).

Teliospores of S. reilianum were collected in August 2020 from naturally infected, smutted panicles of sorghum plants growing in Qianxi county, Bijie City, Guizhou Province, China (Figure 1). Smut galls were broken up and sieved through a 40-mesh sieve to remove plant material and other debris. Teliospores were collected and then air-dried in paper sacks.

Subsequently, 10 mg of teliospores were placed in a 1.5 mL centrifuge tube and mixed well with 1 mL sterile water, followed by centrifugation at 4000 rpm for 2 min at 4°C (ST16R, Thermo Fisher SCIENTIFIC, Braunschweig, Germany) and the supernatant was removed along with any floating debris. The precipitated teliospores were treated with 2% chloramine T for 15 min, followed by centrifugation as described, after which the supernatant was again removed. The teliospores were then washed three times with distilled water with centrifugation between each wash. A small portion of the teliospores was then plated on YEPS medium (2% peptone, 2% sucrose, 1% yeast extract, and 2% agar powder) amended with 30 mg penicillin and 200 mg streptomycin per liter, and the plates were cultured at 28°C under a 12 h light/dark cycle (Martinez et al., 2001). The viability of teliospores was evaluated after 3 d of incubation by monitoring the number of germinated spores out of at least 100 spores in each of four plates. Germination was visually determined using a compound microscope and teliospores were counted as germinated when the length of the germ-tube was longer than one half of the spore diameter. The viability test was repeated three times and the percentage of teliospore germination was calculated based on the collective observations.

Two sets of primers were compared to select a set of appropriate PCR primers which provided the best PCR amplification results using S. reilianum genomic DNA as a substrate. The sequences of the primer pair, YGSP1/GSP2, designed by Ren et al. (2008), are YGSP1 (5′-TCGCCG ACGGATGATAATCG-3′) and GSP2 (5′-GAGTCACCCGCCCAAAGTTA-3). The sequences of the SR3 primer pair, designed by Xu et al. (1999), are SR3-F (5′-GCAGCCTCAGCATTACT C3′) and SR3-R (5′-ATACACCTGTGACGGCTG-3). Genomic DNA was extracted from S. reilianum mycelia, cultured as previously described, using an Ezup column fungal genomic DNA extraction kit (Sangon Biotech Co., Ltd., Shanghai, China), and used as a template for PCR amplification with the designated primers. The primers were synthesized by Biotech Co. Ltd. (Shanghai, China). The reaction system and procedure are listed in Table 1. PCR amplification was conducted in a Bio-Rad C1000 Touch^TM Thermal Cycler (Bio-Rad, Hemel Hempstead, United Kingdom). The amplified products were assessed on a 1% agarose gel using a PowerPac electrophoresis (Bio-rad Laboratories, Inc., United States) unit.

The minimal concentration of fungal DNA needed for amplification using the designed primer pairs was also assessed. The concentration of DNA in the extracted DNA solution was quantified using an ultra-low volume spectrophotometer. In brief, 1 μL of the extracted DNA solution was diluted with 9 μL sterile water, followed by six serial dilutions of 1 in 10. The diluted DNA solutions were then used as a template for PCR amplification with the designated primer pairs. The amplified products were visualized by 1% gel electrophoresis.

Five concentrations (0.15, 0.30, 0.45, 0.60, and 0.75 μg/mL) of tebuconazole were prepared based on a preliminary assay. The level of inhibition determined in the preliminary test at the lowest concentration of tebuconazole was about 10%, while the inhibition level at the highest concentration was about 90%. A stock solution was prepared by dissolving the fungicide in acetone, along with Tween80 as an emulsifier, and then adding water. A total of 50 seeds were soaked in 10 mL of the prepared fungicide solutions for 12 h and then removed and air dried in preparation for subsequent use. Three replicate lots of seed were treated at each fungicide concentration and a solution containing acetone and Tween80, but no fungicide, was used as a control.

The soil used for planting the sorghum was a light sandy clay collected from the teaching farm of Guizhou University and had a pH of 5.7 (H₂O). The soil was autoclaved for sterilization and then sifted for use.

The main factors affecting S. reilianum infection are soil moisture and temperature (Baier and Kruger, 1962; Matyac and Kommedahl, 1985b). Therefore, the incubation conditions were optimized by adjusting the soil moisture, temperature, and spore quantity present in the soil. The optimized protocol for obtaining the highest level of disease incidence was as follows: Soil was mixed with 0.2% (by weight) teliospores and then added to a plug-designed planting tray. Sterilized sorghum seeds were then planted at 2 seeds per well. The seeds were placed 2 cm deep in the pathogen-contaminated soil. The plug trays were then placed in an incubator and cultured at 10°C, 60% humidity, and 20% soil moisture. The trays were then transferred to a greenhouse set at 25°C and a 12 h light/dark cycle. Seedlings were collected after seven days when they were 2–3 cm in length. A total of 18 similar-sized seedlings were collected from each replicate tray and used for the extraction of genomic DNA.

Eighteen seedlings were used within each replicate for DNA extraction. Genomic DNA was extracted from individual seedlings using a DNA extraction kit (Sangon Biotech Co., Ltd., Shanghai, China). Seedling were cleaned of visible soil by rinsing under running tap water and then surface disinfected by submersion in 10% NaOCl solution with agitation for 2 min. Plants were rinsed twice in sterile water for 30 s each time (Anderson et al., 2015). Seedling were placed in a sterilized mortar, ground into a powder with a pestle in liquid nitrogen, and then immediately transferred into a 1.5 mL Eppendorf tube. Next, 800 μL of CTAB extraction buffer solution (preheated in a water bath to 65°C) was added to each of the tubes containing the ground seedling tissues and the mixture was then thoroughly mixed several times on a vortexer (5 min/time) for a total of 20 min. The seedlings were subsequently centrifuged for 2 min (12000 rpm, 4°C). The supernatant was then removed and mixed with an equal volume of phenol: chloroform (1:1, 400 μL each) and the mixture was then centrifuged for 10 min. The resulting supernatant was then mixed with an equal volume of chloroform, followed by centrifugation for 2 min. This process was repeated several times until no protein layer was visible. Genomic DNA was precipitated from supernatant by addition of 2 volumes of ice-cold absolute ethanol and storage at −20°C for 1 h, followed by centrifugation for 2 min. The resulting supernatant was discarded, and the precipitated samples of genomic DNA were washed with 70% ethanol, precipitated twice, dried at room temperature, and finally dissolved in 50 μL of DEPC treated water for subsequent use.

The genomic DNA obtained from sorghum seedlings was used as a template for PCR amplification using the SR3 primers. The reaction system and procedure are provided in Table 1. The PCR products were detected using 1% agarose gel electrophoresis. The incidence of infection of seedlings by the smut pathogen S. reilianum was determined by the analysis of bands present on the agarose gels.

Inhibition of spore germination is a common method to determine the efficacy of fungicides. Therefore, the EC₅₀ and EC₉₅ of tebuconazole were determined by measuring teliospore germination after exposure to the prepared solutions of tebuconazole.

The teliospores were fumigated for 45 min with a mixture of 10 mL/m³ 40% formaldehyde and 5 g/m³ potassium permanganate (Liu et al., 2007). Spore suspensions were prepared with sterile water and adjusted to about 100 spores per field of microscope. A tebuconazole acetone solution was added to sterilized and slightly cooled YEPS medium (pH = 7.0) and fully mixed. Medium amended with an equal amount of acetone was used as a control. Three replicates were used for each concentration of fungicide. Five concentrations of fungicide (0.02, 0.04, 0.08, 0.16, and 0.32 μg/mL) were used based on a preliminary assay. The level of inhibition determined in the preliminary test at the lowest concentration of tebuconazole was about 10%, while the inhibition level at the highest concentration was about 90%. A 0.1 mL drop of spore suspension was evenly distributed on the surface of each YEPS plate (9 cm), and the plates were then placed in an incubator at 28°C (Jiang et al., 2004). The rate of spore germination was determined after 48 h of culture by counting 100 spores in three different fields of view (300 spores per plate). A spore was considered germinated when the length of the germ-tube was longer than one half of the spore diameter.

The level of infection and teliospore germination were calculated using the following equation:

T and CK infection in the seedling assay were determined by a positive PCR amplification in the treated and control seedlings. T and CK in the teliospore germination assay were determined by observations of spore germination in the treated and control teliospores.

The EC₅₀ and EC₉₅ values were calculated by regressing the percentage inhibition (probability value transformed) against the logarithm value of the fungicide concentration (log transformed). Regression analysis was performed using Microsoft Excel 2007 software (Li et al., 2015).

Analysis of Variance between treatments was analyzed using the DPS^® (Data Processing System, version7.05; Hangzhou RuiFeng Information Technology Co. Ltd., Hangzhou, China) software.

Many small colonies of S. reilianum were present on the YEPS medium after incubation for 10 days (Figure 2A). A single colony was selected and cultured on YEPS medium to make observations of colony morphology (Figure 2B). All the morphological observations of colony appearance were consistent with the description of S. reilianum in the Manual of Fungal Identification (Wei, 1979). The percentage of teliospore germination was 67.5%.

The expected band size by SR3 primer was 680 bp. The PCR products amplified by SR3 yielded four clear, distinct bands on an agarose gel (Figure 3). In contrast, only one product was generated by the PCR-amplification using YGSP primers, evidenced as a single band with low definition on an agarose gel. As a result, the SR3 primers were selected for assessment of the presence of S. reilianum, based on the clarity of the bands that were produced by PCR-amplification of S. reilianum genomic DNA.

The concentration of the extracted, genomic DNA of S. reilianum was 107 ng/μL, which was diluted in 10-fold dilution series to obtain concentrations of 10.7 ng/μL, 1.07 ng/μL, 1.07 × 10^–1 ng/μL, 1.07 × 10^–2 ng/μL, 1.07 × 10^–3 ng/μL, and 1.07 × 10^–4 ng/μL. The various concentrations S. reilianum genomic DNA were then used as a template for PCR-amplification with the SR3 primers. As shown in Figure 4, the designated target band was amplified and visible on an agarose gel at concentrations of 10.7 ng/μL, 1.07 ng/μL, and 1.07 × 10^–1 ng/μL of S. reilianum genomic DNA. The band size was approximately 680 bp. The amplification using the 10.7 ng/μL concentration of genomic DNA produced the strongest (brightest) band, while concentrations of 1.07 × 10^–2 ng/μL, 1.07 × 10^–3 ng/μL, and 1.07 × 10^–4 ng/μL did not produce any visible PCR products. Therefore, a concentration of 1.07 ng/μL genomic DNA was considered to be the limit for the detection of S. reilianum.

The incidence of pathogen infection of sorghum seedlings was assessed after seven days of seedling growth. Incidence was determined to be positive if S. reilianum was detected by PCR, while negative PCR results were considered to be a non-infected seedling (Figure 5). Treatment efficacy was determined by the number of negative vs. positive PCR results. The fewer positive PCRs observed, the better the efficacy of the fungicidal treatment.

The incidence of infection in seeds treated with 0.15, 0.30, 0.45, 0.60, and 0.75 μg/mL tebuconazole was 83.33%, 55.56%, 27.78%, 16.67%, respectively, while the incidence of infection in the untreated control was 88.88% (Figure 6). Conversely, the infection inhibition rate was 6.24%, 37.48%, 67.74%, 81.24, and 93.74%, respectively.

A linear regression of the level of inhibition vs. the logarithmic concentration of tebuconazole, yielded the equation y = 6.9494+4.2741x (r = 0.9953). Based on this equation, the EC₅₀ value was calculated to be 0.35 μg/mL tebuconazole (95% confidence limit: 0.33–0.37 μg/mL), and the EC₉₅ was 0.85 μg/mL (95% confidence limit: 0.78–0.92 μg/mL).

The percentage of teliospore germination after 48 h of incubation on media amended with 0.02, 0.04, 0.08, 0.16, and 0.32 μg/mL tebuconazole was 73.58%, 52.44%, 26.83%, 13.00%, and 4.06%, respectively. The germination on media without tebuconazole was 82.00%. Conversely, the teliospore germination inhibition rate was 10.27%, 47.56%, 67.28%, 84.14, and 95.04%, respectively (Figure 7).

Linear regression of the level of inhibition vs. the logarithmic concentration of tebuconazole yielded the equation y = 7.8689+2.2968x (r = 0.9837). Based on this equation, the EC₅₀ value was calculated to be 0.056 μg/mL (95% confidence limit: 0.046–0. 070 μg/mL) and the EC₉₅ was 0.293 μg/mL (95% confidence limit: 0.210–0.410 μg/mL).