Locusts and grasshoppers are major agricultural pests worldwide, causing severe economic and ecological damage during outbreak years and threatening crop production, grassland ecosystems, and rural livelihoods (Lecoq and Zhang 2019, Zhang et al. 2019, Word Ries et al. 2024). Although chemical insecticides remain the principal control method, concerns over their environmental impacts have stimulated the development of microbial biopesticides, including Beauveria bassiana, Metarhizium spp., and Paranosema locustae (Lomer et al. 2001, Lange 2005, Kumar et al. 2021). However, the efficacy of fungal pathogens may be reduced under field conditions because infected locusts and grasshoppers can increase their body temperature through behavioral thermoregulation, thereby inhibiting fungal development (Inglis et al. 1996, Thomas and Jenkins 1997, Fargues et al. 1997, Blanford et al. 1998, Blanford and Thomas 2000).

Aspergillus oryzae is a widely used industrial fungus employed in traditional food fermentations (Barbesgaard et al. 1992, Abe et al. 2006, Zhong et al. 2018). Although species of Aspergillus have rarely been investigated for insect control, A. oryzae XJ-1, isolated from a naturally infected grasshopper in Xinjiang and identified by morphological and molecular analyses, has previously shown pathogenicity against several locust and grasshopper species in laboratory, field-cage, and cropland trials (Zhang et al. 2015, Fu et al. 2024, You et al. 2023). However, its efficacy in natural grasslands has not yet been evaluated.

The Xinjiang Uygur Autonomous Region contains approximately 57 million ha of grasslands inhabited by more than 100 locust and grasshopper species, several of which periodically cause severe damage to rangelands (Chen 1981, Yan et al. 2015). Existing control methods include chemical insecticides (Tong 2015), P. locustae (Wang et al. 1994), Metarhizium flavoviride (Li et al. 2000), and conservation of migratory birds such as Sturnus roseus (Lin 2015). However, few studies have evaluated microbial agents against adult grasshoppers in these arid grassland ecosystems. Because A. oryzae XJ-1 originated from this region, we evaluated its pathogenicity and field efficacy against grasshopper populations through infection assays, field-cage experiments, and field trials.

Field-cage bioassay of the virulence of A. oryzae XJ-1 against adult grasshoppers.—Newly prepared conidial powder of A. oryzae XJ-1 was provided by the Biological Control Laboratory, Institute of Plant Protection, Shandong Academy of Agricultural Sciences. Conidial viability was confirmed by trypan blue staining (Chew et al. 2023). The conidial powder was suspended in 0.3% (v/v) Tween-80 solution at a concentration of 10⁶ viable conidia mL^-1, and conidial density was determined using a hemocytometer. Adult Oedipoda miniata (Pallas, 1771) and Calliptamus barbarus (Costa, 1836) were collected from grasslands in Huocheng County, Yili Kazak Autonomous Prefecture, Xinjiang Uygur Autonomous Region, China. Grasshoppers were maintained outdoors in cages made of steel wire mesh (length: 1 m; width: 1 m; height: 1 m) and fed fresh alfalfa seedings daily. Enclosures were cleaned regularly, and feces were removed daily. The field-cage experiment was conducted at the Yili Prefecture Desert Steppe Experimental Station in Huocheng County. Sixty adults of each species were randomly assigned to six groups—three treatment groups and three control groups—with 10 insects per group. Grasshoppers in the treatment groups were treated with A. oryzae XJ-1 at a concentration of 10⁶ conidia mL^-1, and control insects were treated with 0.3% (v/v) Tween-80 aqueous solution only. For treatments, each adult grasshopper was individually immersed in the conidial suspension for less than 1 s. The insects were briefly air-dried to allow the conidial suspension to adhere to the cuticle and individually transferred to ventilated plastic containers (top diameter: 14 cm; bottom diameter: 9 cm; height: 14 cm). All plastic boxes were placed outdoors on the ground under natural environmental conditions. The experiment was conducted from 20 August to 8 September 2024, during which ambient temperatures ranged from 8 to 32 °C. Mortality was recorded daily for 20 days after treatment.

Large-scale field trials.—Three field trials were conducted. Information on the treated plots and untreated plots are presented in Table 1. Almost all grasshoppers in these plots were adults. No significant rainfall occurred during the three field trials.

An aqueous suspension of A. oryzae XJ-1 conidia at a concentration of 2 × 10⁸ conidia mL^-1 was applied by aerial spraying using a T40 agricultural drone (DJI, Shenzhen, China) at an application rate of 3 × 10¹² conidia ha^-1 in all treatment plots as described by You et al. (2023). Plot 1 and Plot 2 were treated in August of 2024 and July 2025, respectively, and Plot 3 was treated in July of 2025. The untreated plots (CK 1, CK 2, CK 3) did not receive any spray application. Therefore, the present study evaluates the efficacy of the full treatment (conidia + carrier + aerial application) rather than the isolated effect of the fungal conidia alone.

Grasshopper densities were surveyed one day before treatment and on days 5 or 6, 10, and 15 after treatment. Sampling was conducted using a 1-m² quadrat (plastic frame fitted with nylon mesh) placed every 30 steps along a diagonal transect across each plot. Fifty samples were collected from each treatment and control plot during each survey, and the number of grasshoppers within each quadrat was recorded. Population reduction rates were calculated according to the method described by You et al. (2023).

Dead grasshoppers were collected individually from treatment plots to assess fungal colonization. Each cadaver was placed on a sterilized glass slide over sterilized filter paper in a sterile Petri dish. To maintain high humidity, 200 µL of sterile water was added to the filter paper. Petri dishes were incubated at approximately 28 °C for 7 days and examined daily. The presence of A. oryzae XJ-1 yellow conidia growth on the surface of the insect was considered indicative of successful fungal colonization.

Statistical analysis.—All statistical analyses were performed using Origin 8.0 software (OriginLab, Northampton, MA, USA). Student’s t-tests were used to compare cumulative mortality between treatment and control groups for adult O. miniata and C. barbarus in the field-cage bioassay. The same approach was used to compare grasshopper population densities and population reduction rates between treatment and control plots in each field trial. Statistical significance was determined at p < 0.05.

Virulence of A. oryzae XJ-1 against adult grasshoppers in the field-cage experiment.—After being treated with A. oryzae XJ-1 in the field-cage experiment, adult grasshoppers O. miniata and C. barbarus began to die on the second or third day, with mortality gradually increasing thereafter (Fig. 1). By day 20, mortality in treated O. miniata was 73.3 ± 8.8% and that of C. barbarus reached 76.7 ± 6.7%, whereas mortalities in the control groups were 26.7 ± 12.0% and 30.0 ± 11.5%, respectively.

Efficacy of A. oryzae XJ-1 against adult grasshoppers under field conditions.—In Plot 1 (treated in 2024), grasshopper density decreased progressively from one day before treatment to 15 days after application of A. oryzae XJ-1 conidia (Table 2). No significant differences in mean density were observed between treatment and control plots before treatment or at 6 and 10 days after treatment (p > 0.05). However, density was significantly lower in the treatment plot than in the control plot at 15 days after treatment (p < 0.001).

A similar pattern was observed in Plot 2 (treated in 2025). Mean grasshopper density declined after application, with no significant differences between treatment and control plots before treatment or at 5 and 10 days after treatment (p > 0.05). A significant difference was detected at 15 days after treatment (p < 0.001) (Table 2).

In Plot 1, reduction rates were significantly higher in the treatment plot than in the control plot at 10 and 15 days after treatment, reaching 54.8 ± 5.2% and 84.9 ± 4.3%, respectively, compared with 24.9 ± 8.7% and 52.2 ± 7.8% in the control plot (p < 0.05 for both comparisons; Fig. 2). No significant difference was observed at 6 days after treatment (6.0 ± 14.7% versus –15.6 ± 35.0%; p > 0.05).

In Plot 2, reduction rates in the treatment plot were 14.3 ± 0.8%, 43.3 ± 4.2%, and 59.7 ± 4.6% at 5, 10, and 15 days after treatment, respectively. These values were significantly higher than those recorded in the control plot (–13.7 ± 9.8%, 9.8 ± 1.1%, and 2.6 ± 0.6%, respectively; p < 0.05, p < 0.01, and p < 0.001).

In Plot 3, mean grasshopper density declined significantly over time in both treatment and control plots following aerial application of A. oryzae XJ-1 (Table 3). Compared with the control plot, the treatment plot showed significantly lower densities at 5, 10, and 15 days after treatment (p < 0.001, p < 0.001, and p < 0.01, respectively). Reduction rates were significantly higher in the treatment plot at 5 and 15 days after treatment, reaching 79.6 ± 4.8% and 93.2 ± 1.6%, compared with 46.6 ± 4.5% and 79.4 ± 4.0% in the control plot (p < 0.01 and p < 0.05, respectively; Fig. 3). No significant difference was observed at 10 days after treatment (89.7 ± 2.1% versus 54.4 ± 13.7%; p > 0.05).

Mycelial growth of A. oryzae XJ-1 was observed on the cadavers of seven grasshopper species collected from treated plots after 4–5 days of incubation: O. miniata, C. barbarus, S. coerulipes, O. decorus, C. italicus, N. albicornis, and G. sibiricus (Fig. 4).

The effectiveness of fungal biocontrol agents under field conditions is strongly influenced by environmental constraints. Xinjiang grasslands are characterized by low annual precipitation (often <300 mm), high ultraviolet radiation, and a diverse grasshopper community with more than 100 species occurring throughout the growing season (Chen 1981). Drought stress and ultraviolet radiation can reduce the field performance of fungal biopesticides by affecting conidial survival and germination (Braga et al. 2001). Despite these constraints, A. oryzae XJ-1 achieved substantial population reductions against adult grasshoppers under field conditions, suggesting that this indigenous fungal isolate may be well adapted to the environmental conditions of Xinjiang and may have activity against a broad range of grasshopper species.

Several microbial agents have previously been evaluated for grasshopper control in Xinjiang, including Metarhizium spp. and P. locustae. Reported control levels have generally been moderate, with corrected reduction or mortality rates ranging from 37.6% after 14 days for a Metarhizium strain (Mao et al. 2021), to 60.9% after 16 days for an oil formulation of M. flavoviride (Li et al. 2000), and 36.1–56.2% after 28 days for P. locustae (Lin 2015). In comparison, application of A. oryzae XJ-1 resulted in approximately 80% population reduction within 15 days in the present field trials, suggesting promising potential for grasshopper management in arid grassland ecosystems.

The origin of the fungal isolate may contribute to its performance under local conditions. Because A. oryzae XJ-1 was isolated from Xinjiang, its effectiveness may partly reflect adaptation to regional environmental stresses. However, further replicated field experiments are required to confirm its consistency across different habitats and grasshopper assemblages. In addition, the absence of non-target organism assessments in the present study represents an important limitation that should be addressed before large-scale application.

Several factors should also be considered when interpreting the field results. The relatively high reduction rates observed in control plots (particularly CK 1 and CK 3) may have resulted partly from grasshopper movements rather than mortality alone. Adult and nymphal C. italicus and adults of G. sibiricus exhibit strong dispersal abilities, which may substantially influence local density estimates and apparent treatment efficacy at the plot scale (Locust Control Station 1977, Lecoq and Zhang 2019). In addition, mortality recorded in control cages may have been affected by capture stress, variation in adult age, and confinement under outdoor conditions. Because each trial included only one treated plot and one control plot, these experiments should be considered operational demonstrations rather than fully replicated field trials. Future studies should include larger experimental areas, replicated designs, and improved approaches for estimating population-level effects.

Previous studies have shown A. oryzae XJ-1 to be effective against adults of Locusta migratoria, Epacromius spp., Atractomorpha spp., and Oxya spp. under field conditions in Shandong Province at temperatures ranging from 26–38 °C (You et al. 2023). In the present study, field applications were conducted under temperatures ranging from 6–37 °C, suggesting that the operational temperature range of this fungus may be broader than previously reported. However, the mechanisms allowing infection under different thermal conditions remain unclear. In particular, further studies should determine whether infected grasshoppers exhibit behavioral thermoregulation, as reported for M. acridum infections, which may reduce fungal development and affect control efficacy (Inglis et al. 1996, Fargues et al. 1997, Thomas and Jenkins 1997, Blanford et al. 1998, Blanford and Thomas 2000, Bischoff et al. 2009).

In the present study, A. oryzae XJ-1 infection was confirmed in seven grasshopper species—O. miniata, C. barbarus, S. coerulipes, O. decorus, C. italicus, N. albicornis, and G. sibiricus—collected from treated plots. The mechanisms underlying the virulence of A. oryzae XJ-1 remain unknown. Other entomopathogenic fungi produce secondary metabolites involved in pathogenicity, such as destruxins in Metarhizium spp. and beauvericin and related compounds in B. bassiana (Gillespie and Claydon 1989, Wang et al. 2021, Bi et al. 2023). Similar mechanisms may contribute to the activity of A. oryzae XJ-1, but identification of potential toxins and characterization of infection pathways require further investigation. Future trials should also include carrier-only controls to improve interpretation of formulation effects.

In conclusion, A. oryzae XJ-1 reduced grasshopper populations and infected multiple grasshopper species under field conditions in the arid grasslands of Xinjiang. Although additional replicated trials, formulation studies, and evaluations of non-target effects are required, this locally isolated fungus shows promising potential as a biological control agent for grasshopper management in northwestern China.