S. furcifera was collected from a rice field in Hongyanba (105.4756° E, 28.1880° N), Xuyong County, Luzhou City, Sichuan Province, China, in 2020. It was then reared on rice (TN1) seedlings (approximately 15 cm tall) in the intelligent artificial climate chamber (RXE-450, Ningbo Jiangnan Instrument Factory Co., Ltd., Ningbo, China) for over 15 generations without exposure to any pesticides. The chamber was maintained at 26 ± 1°C, with a relative humidity of 70 ± 10%, and a photoperiod of 14 h: 10 h (L:D).

Test strains (Specific information is provided in Table 1). The B. bassiana strain BEdy1 used in the test was provided by Shang Jing’s research group from the Phytopathology Laboratory, College of Agronomy, Sichuan Agricultural University. BEdy1 (GenBank: MK345993) was isolated from the body of the soybean pest, Ergania Doriae Yunnanus, collected from a soybean field in Renshou County, Sichuan Province, in September 2018. A BLAST comparison of the 544 bp rDNA-ITS of the strain revealed that BEdy1 shared 99% similarity with strain JEF006 (GenBank: KT280276). Combining these results with morphological observations of the strain, the pathogen causing field mortality of Ergania Doriae Yunnanus was ultimately identified as B. bassiana BEdy1 (Zhang et al., 2020). The B. bassiana JZ21004 is a commercial strain from Hubei QIMING BIO Engineering Co., Ltd. The remaining six B. bassiana were isolated and identified from muscardine cadavers collected in the field. These test strains were cultured on PPDA medium (consisting of 200 g potato, 20 g dextrose, 20 g agar, and 10 g peptone) in plastic Petri dishes (90 mm diameter), which were then wrapped with Parafilm and placed in darkness at 26 ± 1°C. Each strain was inoculated onto slant culture medium and stored at 4°C for future use, with additional preservation in 50% glycerol at −80°C in an ultra-low temperature freezer.

Comparison of colony growth rates (Li et al., 2014). A 5 mm diameter fungal plug was taken from the reserved B. bassiana culture medium and placed at the center of a new culture medium. The cultures were then incubated in a dark incubator at 26°C with three replicates set up for each strain. On the 3rd and 7th days, the diameters of the colonies were measured in both vertical and horizontal directions, and their average values were taken to calculate the daily average growth rate of the colonies (mm/d).

Comparison of initial sporulation time and sporulation yield (Li et al., 2014). After the mycelium grew on the culture medium, a small amount of mycelium was collected daily using a colorless, transparent adhesive tape. Under the microscope, we observed the presence of conidia and recorded the initial sporulation time. After 15 days of cultivation, six 6 mm diameter fungal plugs were taken from the midpoint between the center and the edge of the colony in different directions and placed into 10 mL centrifuge tubes containing 5 mL of 0.05% Tween-80 sterile aqueous solution. The tubes were then shaken on a thermostatic oscillator for 30 min to obtain a spore suspension. Subsequently, the spore content in the spore suspension was observed under a microscope using a hemocytometer and converted to the number of spores per cm² of colony. Each strain was tested in triplicate, and the average values were taken.

Comparison of spore germination rates (Li et al., 2014). After thoroughly washing the conidia on the PPDA plate with 0.05% Tween-80 solution, a spore suspension of 1.0 × 10⁷ spores/mL was prepared and incubated in a constant temperature oscillator at 25°C and 150 rpm for 24 h. The spore germination of each strain (spore tube larger than the short radius of the spore is considered germination) was observed using a blood cell counting plate, and the spore germination rate was calculated by examining five different fields of view under a microscope. Each strain was set to four replicates and the average value was calculated.

The spray method was used to screen highly virulent strains against 3rd instar nymphs of WBPH (Lin et al., 2013). All eight strains were prepared into spore suspensions at a concentration of 1 × 10⁸ spores/mL. Rice seedlings at the 4–5 leaf stage were washed with clean water, dried, and then grouped into sets of 20 plants. Each set was inoculated with 30 third-instar nymphs in the mid-stage. A handheld sprayer was used to uniformly apply 3 mL of the spore suspension. A 0.05% Tween-80 solution served as the control. The seedlings were then placed in an artificial climate chamber (T = 27°C ± 1°C, RH = 75% ± 5%, L: D = 14: 10 h) for rearing, and the rice seedlings were replaced every 3 days. The number of dead WBPH in each treatment was counted daily within 10 days (gently touching the insect body with a brush, using the criteria of weakness and inability to crawl normally as the standard for death). The dead insect bodies were maintained under moist conditions, and fungi were isolated and identified from their body surface and hemocoel, respectively. Finally, the number of mummified insects was recorded. Both the treatments and the control were set up in triplicate.

Retesting of the screened strains with the highest pathogenicity. The concentration gradient of spore suspension is set as follows: 1.0 × 10⁵ spores/mL, 1.0 × 10⁶ spores/mL, 1.0 × 10⁷ spores/mL, 1.0 × 10⁸ spores/mL, and 1.0 × 10⁹ spores/mL. Using 0.05% Tween-80 solution as a control, the number of deaths from WBPH was counted daily within 10 days of spraying. The treatment and control groups were set to three replicates.

Fluorescence staining was used to observe the invasion process of B. bassiana in the nymphs of WBPH (Wang et al., 2015). 40 mg of fluorescein dye FDA (Solarbio Company) was dissolved in 10 mL of acetone to form an FDA stock solution. 88 μL of the FDA stock solution was then diluted in 10 mL of deionized water to prepare the FDA working solution, which was stored in a dark, cold place for later use. The inoculation method was the same as described in section 2.3. Samples were collected at 12 h, 24 h, 36 h, 60 h, 84 h, and 108 h post-inoculation, with one test insect selected at each time point. Before observation, the samples were placed on clean slides and treated with the FDA working solution, allowing them to react in the dark for 5 min. Subsequently, the infection status of nymphs by B. bassiana BEdy1 at each time point was observed using a fluorescence microscope with a GFP filter, and photographs were taken for recording.

The method of Toledo et al. (2010) was adopted with slight modifications. The inoculation procedure was the same as described in section 2.3. Samples were collected at 12 h, 24 h, 72 h, 96 h, and 120 h post-inoculation, with one test insect selected at each time point. Sterile water with 0.05% Tween-80 served as the control. After sampling, the samples were fixed in 2.5% glutaraldehyde at 4°C for 24 h and then washed three times with 0.1 mol/L phosphate buffer for 10 min each time to ensure complete removal of the fixative. Subsequently, the samples were fixed in 1% osmic acid for 2 h and then dehydrated in a graded series of 30, 50, 70, 90, and 100% ethanol for 15 min each. After dehydration, the samples were air-dried at room temperature for 2 h. They were then mounted on a scanning electron microscope (SEM) stub using conductive adhesive tape, vacuumed, and coated with gold. Observations were made under the SEM at appropriate positions and magnifications, and photographs were taken for recording.

The method of Toledo et al. (2010) was adopted with slight modifications. The inoculation procedure was the same as described in section 2.3. Samples were collected at 60 h, 72 h, and 96 h post-inoculation, with one test insect selected at each time point. Sterile water with 0.05% Tween-80 served as the control. The samples were fixed in Bouins fixative at 4°C for 24 h, followed by graded dehydration in 35, 50, 70, 80, 90, 95, and 100% ethanol for 1 h each. Subsequently, the samples were infiltrated in 50, 70, 85, and 100% xylene-ethanol solutions and 50% paraffin-xylene for 1 h each, and then immersed in paraffin at 60°C for 2 h. After tissue embedding, sections of the thorax and abdomen of the insect were cut using a rotary microtome with a thickness of 8 μm. Following spreading in a 35°C water bath, the sections were picked up using adhesive slides, dried in a 37°C oven, and stained according to the instructions of the Hematoxylin and Eosin (HE) staining kit. They were then dehydrated in a series of ethanol concentrations, mounted with neutral balsam, and dried at 37°C. Subsequently, histopathological changes in the WBPH infected by B. bassiana were observed under an optical microscope, and photographs were taken for recording.

The average growth rate, initial spore production time, spore yield, spore germination rate, cumulative corrected mortality, and muscardine cadaver rates of the strains were analyzed for significant differences using SPSS 22.0. Duncan’s new multiple range method was used for multiple comparisons of the means. Using the Probit method of POLO 2.0 software to calculate the LT₅₀ and LC₅₀. The line chart was drawn using Graphpad Prism 9.5 software. The formula used for pathogenicity testing is as follows: Mortality % = Number of live insects before treatment − Number of live insects after treatment Number of live insects before treatment × 100 Corrected mortality % = Mortality of the treatment group − Mortality of the control group 1 − Mortality of the control group × 100 Muscardine cadaver rate % = Number of muscardine cadavers Number of tested insects × 100

Through observation, it was found that the surface of most colonies appeared white or milky white, while a few were powdery white or yellowish-white; the reverse side was mostly light yellow (Table 2). Additionally, some colonies exhibited concentric patterns or groove-like lines. Based on the appearance of different B. bassiana on PPDA medium, they can be roughly classified into three types: powdery, flocculent, and tomentose colonies (Table 2). The strains BEdy1, JZ21004, and WJGP15 are spore-forming strains with thin, powdery colonies and high spore production (Table 2). The mycelial strains (WJGP3, CZ9) have flocculent colony morphology and produce fewer spores (Table 2). The colonies of WJGP1, WJGP5, and WJGP11 are tomentose, with white or milky white spore powder. Among them, WJGP11 has radial folds and aerial hyphae appear in the later stages (Table 2).

The vegetative growth rate of strains is one of the indicators reflecting their excellent characteristics. Vegetative growth measurements were conducted for eight strains, and the results showed significant differences in colony growth rates among the different test strains (Table 3). Among them, the BEdy1, WJGP1, and WJGP11 strains grew faster, with a daily average increase in colony diameter greater than 3.3 mm, which was significantly higher than that of the WJGP3, WJGP5, CZ9, and WJGP15 strains (p < 0.05).

The time required for the WJGP1 strain to start sporulation (4.67 days) was significantly shorter than that of the WJGP3, WJGP5, CZ9, WJGP11, and WJGP15 strains (p < 0.05), and was 3 days earlier than the latest sporulation producing CZ9 strain (7.67 days), but there was no significant difference compared to the BEdy1 and JZ21004 strains (p > 0.05). In addition, there were significant differences in spore production among different strains, with BEdy1, JZ21004, and WJGP15 strains producing significantly more spores than the other tested strains (p < 0.05). Among them, BEdy1 strain had the highest spore production, reaching 1.37 × 10⁸ spores/cm², which was 9.26 times the spore production of loose hyphal CZ9 strain (1.48 × 10⁷ spores/cm²).

The germination rate of spores is also an indicator of the vitality of strains. The results showed that there were significant differences in the germination rates of spores among the different strains (Table 3). The BEdy1 strain had a germination rate of 96% at 24 h, which was significantly higher than that of other strains (p < 0.05). Based on the comprehensively measured indicators, the spore production and spore germination rate of the BEdy1 strain were significantly higher than those of the other strains, and the vegetative growth was faster, demonstrating excellent biological characteristics.

In order to compare the pathogenic effects of different B. bassiana on WBPH, we determined the pathogenicity of eight B. bassiana strains towards the insect. Experimental results revealed that there were significant differences (p < 0.05) in the pathogenicity of different B. bassiana strains against the third-instar nymphs of WBPH (Table 4). The cumulative corrected mortality of the WBPH was 54.06% ~ 77.67%, the muscardine cadaver rate was 31.11% ~ 57.78%, and the LT₅₀ value was 5.12 ~ 8.07 days. After 10 days of treatment, there were three WBPH strains with a cumulative corrected mortality of over 70%, namely BEdy1, WJGP15, and JZ21004. Among them, the BEdy1 strain showed the highest cumulative corrected mortality against WBPH (p < 0.05), reaching 77.67% with an LT50 value of 5.12 days, demonstrating the highest pathogenicity. At the same time, after spraying with a high-concentration spore solution, muscardine cadavers appeared in all the strains. The muscardine cadaver rates of the BEdy1 and WJGP15 strains were 57.78 and 54.44%, respectively, which were significantly higher than those of the WJGP1 (44.44%), WJGP3 (38.89%), and CZ9 (26.67%) strains (p < 0.05). In summary, among the eight tested strains, the BEdy1 strain showed strong pathogenicity against the 3rd instar nymphs of the WBPH.

Furthermore, we conducted a detailed investigation into the pathogenicity of B. bassiana BEdy1 towards the WBPH. As shown in Figure 1, the cumulative corrected mortality of the 3rd instar nymphs of the WBPH was positively correlated with spore suspension concentration and treatment time of the B. bassiana BEdy1 strain. When treated with high concentrations of 1.0 × 10⁹ and 1.0 × 10⁸ spores/mL, a large number of WBPH died from the second day, and the cumulative mortality of the test insects after 10 days reached 83.54 and 77.67%, respectively; When treated with concentrations of 1.0 × 10⁵, 1.0 × 10⁶ and 1.0 × 10⁷ spores/mL, the mortality of the test insects increased significantly from the 3rd day, and the cumulative corrected mortality rates on the 10th day were 58.83, 45.85 and 28.20%, respectively. The pathogenicity of the B. bassiana BEdy1 strain to WBPH exhibits a time-dose relationship (Table 5), meaning that the higher the spore concentration of the treated insect, the smaller the LT₅₀ value. Among them, the lethal time under the treatment of 1.0 × 10⁹ and 1.0 × 10⁸ spores/mL concentrations was 4.432 and 4.932 days, respectively. However, when the spore suspension concentration was between 1.0 × 10⁵ and 1.0 × 10⁷ spores/mL, the LT₅₀ value of the test insect exceeded 6 days. Meanwhile, the LC₅₀ value (10 days) of B. bassiana BEdy1 against the third-instar nymphs of WBPH was calculated to be 2.13 × 10⁶ spores/mL, with a 95% confidence interval of 8.81 × 10⁵ to 4.43 × 10⁶ spores/mL.

After inoculation with BEdy1 strain for 12 h, a large number of viable spores aggregated near the abdominal sensors on the body surface (Figure 2A). After attaching to the body wall, the conidia were observed to start germinating into germ tubes at 24 h, with a small number germinating into hyphae (Figure 2B). At 36 h post-inoculation, the germinated hyphae extended horizontally on the insect’s surface, seeking suitable sites to penetrate the insect’s body wall (Figure 2C). By 60 h post-inoculation, some hyphae had proliferated within the insect body (Figure 2D). At 84 h post-inoculation, a large number of hyphae aggregated and grew at the abdominal end (Figure 2E), and a significant number of WBPH died at this stage. At 108 h post-inoculation, the hyphae had broken through the body wall and grown extensively on the insect’s head, including the compound eyes, vertex, and gena (Figure 2F).

The scanning electron microscopy observation results are shown in Figure 3. Twelve hours after inoculation, the conidia were successfully attached to the small eyes and bristles near the head of the insect (Figures 3A,B). After 24 h of inoculation, a large number of conidia gathered in the abdomen of the insect and some spores had grown hyphae (Figure 3C). After 72 h after inoculation, the hyphae that penetrated the body wall were wound parallel to the compound eye (Figure 3D). After 96 h of inoculation, the mycelia grew and spread extensively in the abdomen of the insect (Figure 3E). After 120 h of inoculation, the head compound eye and bristles were extensively covered by the hyphae and conidia of B. bassiana, and the hyphae were intertwined to form a network structure (Figures 3F,G). At the same time, newly generated conidia were also observed to attach to the body wall, indicating that a suitable host is conducive to fungal survival and can promote the re-infection cycle of B. bassiana. In addition, the body wall of the uninfected WBPH was smoother (Figure 3H).

Tissue observation results at different time periods after inoculation are shown in Figure 4. After 60 h of inoculation, mycelial segments were observed in the internal tissues and organs of infected insects. The neuropile of the infected insect began to shrink, the trachea broke, and the surrounding muscle tissue appeared as loose strips (Figure 4A). After 72 h after inoculation, a large number of hyphae appeared in the hemocoel of the infected nymphs, with a loose arrangement of body wall muscles. The epidermis and internal structure of the nymphs were separated, and the hyphae grew outward and accumulated on the surface of the nymphs (Figure 4B), indicating that B. bassiana grew rapidly inside the nymphs. After 96 h of inoculation, many hyphae invaded the digestive tract and surrounded the dorsal blood vessels. The infected insect tissue became blurry and structurally incomplete, suggesting that it was the result of mechanical pressure generated by the hyphae or secretion of hydrolytic enzymes (Figure 4C). The tissue structure of the control insects was complete, and the muscle and other tissues were clearly visible (Figure 4D).