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Brief Report

Synergistic Effect of Mixture of Microsporidium Nosema locustae (Protista: Microsporidia) and Novel Fungus Aspergillus oryzae XJ-1 (Eurotiales: Trichocomaceae) Against Adult Locusta migratoria (Orthoptera: Acrididae) in Laboratory

by
Pengfei Zhang
1,2,†,
Mingquan Yang
3,†,
Yinwei You
1,* and
Long Zhang
1,*
1
Shandong Key Laboratory for Green Prevention and Control of Agricultural Pests, Institute of Plant Protection, Shandong Academy of Agricultural Sciences, Jinan 250100, China
2
Hebei Provincial Jujube Kernel Utilization Technology Innovation Center, Department of Chemical Engineering and Biotechnology, Xingtai University, Xingtai 054001, China
3
Department of Grassland Resources and Ecology, College of Grassland Science and Technology, China Agricultural University, Beijing 100193, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Agronomy 2025, 15(2), 364; https://doi.org/10.3390/agronomy15020364
Submission received: 30 December 2024 / Revised: 23 January 2025 / Accepted: 28 January 2025 / Published: 30 January 2025
(This article belongs to the Section Pest and Disease Management)
Browse Figure
Versions Notes

Abstract

:
Locust adults can form gregarious swarms and cause locust plagues. Few studies have evaluated the efficacy of a mixture of two biocontrol agents for controlling locust adults. Here, we assessed the effects of the mixture of a protozoan biocontrol agent, Nsoema locustae, and a fungal agent, Aspergillus oryzae XJ-1, at two ratios against locust adults in the lab. Synergistic effects of the mixture were observed (χ2 > χ2 (df, 0.05) and Po > PE). The maximum mortality caused by an N. locustae–A. oryzae XJ-1 mixture was 92.67% on the 12th day after inoculation, much higher than those of each agent. In addition, the median survival times were significantly lower when locusts were exposed to the mixture than when they were exposed to N. locustae or A. oryzae XJ-1 alone (p < 0.05).

1. Introduction

Locusta migratoria are widely distributed in Asia, Africa, the Americas, and Australia, and they induce severe damage to thousands of acres of crops and rangelands annually [1]. Swarms of adult locusts can lead to plague outbreaks because of their ferocious appetites and strong dispersal ability [1,2]. L. migratoria mostly occur in remote and desolate areas where the ability to monitor pests in a timely and accurate manner is limited, and this makes implementing control strategies that target locusts in the early developmental stages a major challenge. Artificial chemical insecticides are often used to control locusts in the adult stage, but these insecticides have deleterious effects on the environment and non-target organisms; there is thus a need to develop a biocontrol agent that can effectively control L. migratoria in the adult stage.
The microsporidium Nosema locustae ([=Paranosema locustae], [=Antonospora locustae]), is a pathogen of locusts and grasshoppers (Insecta, Acrididae), and it mainly infects the fat body tissue [3,4]. N. locustae was the first protozoan insecticide to be commercially developed in world [5,6,7]. Up to now, 144 species of Orthopteran insects are susceptible to N. locustae; however, it is innocuous to non-orthopteran insects and other non-target animals [8,9,10]. It is transmitted orally and is, therefore, probably less sensitive to environmental factors than fungal-type agents. This is the reason why it proved successful at a 3000–4000 m altitude on the Tibetan plateau in China and in high-temperature regions, such as Hainan province in China, Lao, and Vietnam [8,11,12]. The long-term epizootics of N. locustae have been investigated in China and Argentina, which showed that this pathogen can persist for many years after its introduction [13,14,15]. The relatively slow action of N. locustae in killing locusts, especially adult locusts, is its major drawback [11]. For the migratory locust L. migratoria migratorioides (Reiche & Fairmaire, 1849), the younger the nymph, the more susceptible to infection they were found to be [6]. Similar results were obtained in the desert locust Schistocerca gregaria (Forskål, 1775) and the Senegalese grasshopper Oedaleus senegalensis (Krauss, 1877), and the median survival times of infected 1st, 3rd, and 5th instars of O. senegalensis were 5, 9, and 15 days, respectively, when the locusts were inoculated with 1 × 107 spores [16]. Consequently, N. locustae has generally been applied when locusts and grasshoppers are nymphs. The same strategy has also been used when applying Metarhizium spp. for locust and grasshopper control.
A novel fungus originally isolated from dead locusts collected in Xinjiang Uyghur autonomous region, China, was identified as Aspergillus oryzae XJ-1 [17,18,19]. Previous studies conducted by our lab have shown that A. oryzae XJ-1 was pathogenic to both the nymph and adult stages of L. migratoria [17,18,19].
A suitable mixture of Nosema and fungus or virus pathogens can show synergistic effects on target pests. N. ceranae could accelerate deformed wing virus (DWV) replication in infected bees in a dose-dependent manner in the early stages of DWV infection [20]. LC50 values of Bacillus thuringiensis against European corn borers, Ostrinia nubilalis, for N. pyrausta-infected larvae were significantly lower than for uninfected larvae [21]. Some studies on the impact of N. locustae and Metarhizium anisopliae (a widely used fugal pathogen of locusts) mixtures against locust nymphs have been conducted. Fifth-instar desert locust (S. gregaria Forskål) nymphs treated with both N. locustae and M. anisopliae died sooner than those infected with only one of the pathogens [22]. A synergistic effect was also observed in nymphs of the migratory locust Melanoplus sanguinipes treated with a combination of N. locustae and M. anisopliae [23]. In this study, we attempted to explore the effects of a combination of N. locustae and A. oryzae XJ-1 against adult L. migratoria and to provide a new biological control strategy for L. migratoria.

2. Materials and Methods

2.1. Insects and Pathogens

Cultures of L. migratoria and its pathogens N. locustae and A. oryzae XJ-1, as well as the spore preparations of the two pathogens, were maintained as described previously [17,24]. The test locusts were adult locusts that had emerged 2–8 days previously and were reared with enough wheat leaves to survive in an incubator at a constant temperature of 30 ± 1 °C, 70 ± 5% relative humidity, and an L18:D6 photoperiod throughout the experiment. N. locustae and A. oryzae XJ-1 were diluted to the same concentration of 2 × 107 spores/mL with 0.1% Tween 80. A 1:1 mixture (M1:1) and 1:2 mixture (M1:2) (v/v) were prepared by directly mixing spore suspensions of N. locustae (2 × 107 spores/mL) and A. oryzae XJ-1 (2 × 107 spores/mL) prior to each bioassay.

2.2. Bioassays

With reference to the method used to spray pesticides in fields, test locusts reared with sparse potted wheat (8 cm per side of the square bottom, 6 cm and 10 cm in height of the pot and wheat, respectively) in 4500 mL plastic boxes (15 cm per side of the square bottom, 20 cm in height) were liberally sprayed with pathogen suspension for bioassays. The test locusts reared with sparse potted wheat from different treatments (N. locustae alone, A. oryzae XJ-1 alone, M1:1 and M1:2) were sprayed with the corresponding pathogen suspension (N. locustae suspension of 2 × 107 spores/mL, A. oryzae XJ-1 suspension of 2 × 107 spores/mL, 1:1 mixture and 1:2 mixture (v/v) of the two-pathogen suspension) using a sterile sprinkling bottle in the same way, ensuring the droplets landed on the surface of the locusts and wheat leaves. Each plastic box contained 1 potted wheat and 3 locusts, and a total of 30 locusts were tested for each replicate. A period of 3 days later, the locusts were individually placed into smaller plastic boxes (10 cm per side of the square bottom, 15 cm in height), and treated wheat leaves were changed for fresh wheat leaves for normal feeding. Control insects were sprayed with Tween 80 (sigma, Saint Louis, MO, USA), and the other treatments were the same as the treatment group. Fresh wheat leaves were provided daily, and feces were removed daily. Locust mortality was checked daily for 21 days. Two sets of experiments were conducted. One set of experiments comprised treatments with N. locustae, A. oryzae XJ-1, M1:1, and the corresponding controls, with three replicates of each treatment (30 insects/replicate); the other set of experiments comprised treatments with N. locustae, A. oryzae XJ-1, M1:2, and the corresponding controls, with six replicates of each treatment (30 insects/replicate).

2.3. Statistical Analyses

Analyses for additive, antagonistic, and synergistic interactions were based on a binominal test which involved comparing the expected and observed mortalities [22]. The expected mortality at a range of concentrations of N. locustae and A. oryzae XJ-1 is based on the formula PE = Pc + (1 − Pc)P1 + (1 − Pc)(1 − P1)P2, in which PE is the mortality expected from the combination of the two pathogens, Pc is the natural (control) mortality, P1 is the mortality from N. locustae alone, and P2 is the mortality from A. oryzae XJ-1 alone. The chi-square values were based on the formula χ2 = (Lo − LE)2/LE + (Do − DE)2/DE in which Lo is the observed number of living larvae, LE is the expected number, Do is the observed number of dead nymphs, and DE is the expected number. This formula was used to test the hypothesis of independence (df, p = 0.05). Additivity was indicated if χ2 < χ2 (df = 1, 0.05). Antagonism was indicated if χ2 > χ2 (df = 1, 0.05) and Po < PE. Synergism was indicated if χ2 > χ2 (df = 1, 0.05) and Po > PE. Po and PE represent the observed and expected mortality, respectively, from the combination of the two pathogens.
Median survival times (MSTs) were estimated using the Kaplan–Meier procedure followed by a log rank test. The statistical analyses were performed using IBM SPSS statistics 20.0 software (IBM, Armonk, NY, USA).

3. Results

In general, the lethal effects of N. locustae, A. oryzae XJ-1, and the mixtures on locust adults increased with time within the 21 observation days. The mortality induced by N. locustae increased gradually after day 5. The cumulative mortality rate for the mixture and A. oryzae groups increased rapidly following the 3rd day after inoculation and peaked on the 9th to 13th days post inoculation (Figure 1). In the 1:1 mixed infection assay, five of seven (from the onset of mortality to day 9) mixed treatments of the two pathogens in observed mortality produced synergistic effects (χ2 > χ2 (df = 1, 0.05) = 3.84 and Po > PE) in which 86.67% of the adult locusts were killed, as compared to the expected mortality (Figure 1).
In the 1:2 mixed infection assay, six of ten (from the onset of mortality to day 12) mixed treatments of the two pathogens in observed mortality produced synergistic effects (χ2 > 3.84 and Po > PE) in which 92.67% of the adult locusts were killed, as compared to the expected mortality (Figure 1). In addition, the locust median survival times (MSTs) were significantly lower in the mixed treatment of M1:1 (7.00 ± 0.38 days) than in the treatments with N. locustae (15.00 ± 0.91 days, p < 0.001) and A. oryzae XJ-1 (9.00 ± 0.63 days, p = 0.02 < 0.05) alone (Table 1). MST was also observed to be significantly lower in the mixed treatment of M1:2 (7.00 ± 0.27 days) than in the treatments with N. locustae (16.00 ± 1.61 days, p < 0.001) and A. oryzae XJ-1 (9.00 ± 0.55 days, p < 0.001) alone (Table 1).

4. Discussion

The findings of our study indicated that the mixture of microsporidium N. locustae and the fungal pathogen A. oryzae in observed mortality produced synergistic effects in the early stage of application as compared to the expected mortality. These findings are consistent with the results of Tokarev et al. and Tounou et al., showing that locusts died sooner and the observed mortality was higher as compared to the results expected from the combination, when N. locustae and Metarhizium spp. were applied combinedly [22,25]. In addition, the synergistic effects of N. locustae and A. oryzae against L. migratoria should also be verified in the field, and this will determine whether the mixture of the two pathogens can be formulated as a biological control agent in future. The interaction mechanism of the mixture was not very clear. The synergistic effect from the two pathogens results from interspecific interactions as N. locustae has a debilitating effect on its locust hosts by infecting the fat body tissue and disrupting intermediary metabolism [26]. In addition, Lv et al. found that the expression levels of defensin genes in the fat body of migratory locusts after N. locustae infection fell to the lowest over the course of 10 days [27]. Synergistic mechanisms from the two pathogens can be explored from these perspectives in the future.

Author Contributions

Conceptualization, L.Z. and P.Z.; methodology, M.Y. and L.Z.; experiment performance, M.Y. and P.Z.; writing—review and editing, P.Z., L.Z. and Y.Y.; supervision, L.Z. and Y.Y. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the Agricultural scientific and technological innovation project of the Shandong Academy of Agricultural Sciences (CXGC2024F05).

Data Availability Statement

Data are contained within the article.

Acknowledgments

Authors would like to thank anonymous reviewers’ comments and suggestions.

Conflicts of Interest

The authors declare no conflicts of interest.

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Agronomy 15 00364 g001
Figure 1. Cumulative mortality curves of adult L. migratoria after exposure to the treatments of Nosema locustae, Aspergillus oryzae XJ-1, Mixture 1:1 (M1:1), and Mixture 1:2 (M1:2). Nosema locustae: locusts were infected with an N. locustae suspension at 2 × 107 spores/mL; Aspergillus oryzae XJ-1: locusts were infected with an A. oryzae XJ-1 suspension of 2 × 107 spores/mL; Mixture 1:1 (observed): locusts were infected with a mixture of N. locustae (2 × 107 spores/mL) and A. oryzae XJ-1 (2 × 107 spores/mL) at a ratio of 1:1 (v/v); Mixture 1:1 (expected): the mortality expected from the combination of the two pathogens at a ratio of 1:1 (v/v); Mixture 1:2 (observed): locusts were infected with a mixture of N. locustae (2 × 107 spores/mL) and A. oryzae XJ-1 (2 × 107 spores/mL) at a ratio of 1:2 (v/v); Mixture 1:2 (expected): the mortality expected from the combination of the two pathogens at a ratio of 1:2 (v/v); Control: locusts were sprayed with Tween 80. The expected mortality at a range of concentrations of N. locustae and A. oryzae XJ-1 is based on the formula PE = Pc + (1 − Pc)(P1) + (1 − Pc)(1 − P1)(P2), in which PE is the mortality expected from the combination of the two pathogens, Pc is the natural (control) mortality, P1 is the mortality from N. locustae alone, and P2 is the mortality from A. oryzae XJ-1 alone. “S” (the abbreviation of synergism) indicated that mixed treatments of the two pathogens in observed mortality produced synergistic effects (χ2 > χ2 (df, 0.05) and Po > PE), as compared to the expected mortality. Po represents the observed mortality from the combination of the two pathogens. The chi-square values (χ2) were based on the formula χ2 = (Lo − LE)2/LE + (Do − DE)2/DE in which Lo is the observed number of living larvae, LE is the expected number, Do is the observed number of dead nymphs, and DE is the expected number. Po represents the observed number from the combination of the two pathogens. Data represent the mean ± S.E of 3 or 6 replicates, and each replicate contained 30 adult locusts 2–8 days after emergence.
Figure 1. Cumulative mortality curves of adult L. migratoria after exposure to the treatments of Nosema locustae, Aspergillus oryzae XJ-1, Mixture 1:1 (M1:1), and Mixture 1:2 (M1:2). Nosema locustae: locusts were infected with an N. locustae suspension at 2 × 107 spores/mL; Aspergillus oryzae XJ-1: locusts were infected with an A. oryzae XJ-1 suspension of 2 × 107 spores/mL; Mixture 1:1 (observed): locusts were infected with a mixture of N. locustae (2 × 107 spores/mL) and A. oryzae XJ-1 (2 × 107 spores/mL) at a ratio of 1:1 (v/v); Mixture 1:1 (expected): the mortality expected from the combination of the two pathogens at a ratio of 1:1 (v/v); Mixture 1:2 (observed): locusts were infected with a mixture of N. locustae (2 × 107 spores/mL) and A. oryzae XJ-1 (2 × 107 spores/mL) at a ratio of 1:2 (v/v); Mixture 1:2 (expected): the mortality expected from the combination of the two pathogens at a ratio of 1:2 (v/v); Control: locusts were sprayed with Tween 80. The expected mortality at a range of concentrations of N. locustae and A. oryzae XJ-1 is based on the formula PE = Pc + (1 − Pc)(P1) + (1 − Pc)(1 − P1)(P2), in which PE is the mortality expected from the combination of the two pathogens, Pc is the natural (control) mortality, P1 is the mortality from N. locustae alone, and P2 is the mortality from A. oryzae XJ-1 alone. “S” (the abbreviation of synergism) indicated that mixed treatments of the two pathogens in observed mortality produced synergistic effects (χ2 > χ2 (df, 0.05) and Po > PE), as compared to the expected mortality. Po represents the observed mortality from the combination of the two pathogens. The chi-square values (χ2) were based on the formula χ2 = (Lo − LE)2/LE + (Do − DE)2/DE in which Lo is the observed number of living larvae, LE is the expected number, Do is the observed number of dead nymphs, and DE is the expected number. Po represents the observed number from the combination of the two pathogens. Data represent the mean ± S.E of 3 or 6 replicates, and each replicate contained 30 adult locusts 2–8 days after emergence.
Agronomy 15 00364 g001
Table 1. Median survival times (MSTs) of adult locusts of Locusta migratoria treated with different treatments and MST comparisons between each other.
Table 1. Median survival times (MSTs) of adult locusts of Locusta migratoria treated with different treatments and MST comparisons between each other.
TreatmentsMST (95% FL) aMST Comparison
Nosema (1:1)Aspergillus (1:1)Nosema (1:2)Aspergillus (1:2)
χ2Sig. bχ2Sig.χ2Sig.χ2Sig.
Nosema (1:1)15.00 ± 0.91 (13.22–16.79) 16.610.00
Aspergillus (1:1)9.00 ± 0.63 (7.76–10.24)
Mixture (1:1)7.00 ± 0.38 (6.26–7.74)40.370.005.950.02
Nosema (1:2)16.00 ± 1.61 (12.85–19.15) 23.940.00
Aspergillus (1:2)9.00 ± 0.55 (7.93–10.07)
Mixture (1:2)7.00 ± 0.27 (6.47–7.53) 86.930.0017.900.00
a Kaplan–Meier Survivorship analysis was used to calculate the MST; 95% FL: 95% fiducial limits. b Sig.: significance; χ2 > χ2 (df, 0.05) indicate that the comparison results for the two treatments were significantly different (Sig. < 0.05).
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Zhang, P.; Yang, M.; You, Y.; Zhang, L. Synergistic Effect of Mixture of Microsporidium Nosema locustae (Protista: Microsporidia) and Novel Fungus Aspergillus oryzae XJ-1 (Eurotiales: Trichocomaceae) Against Adult Locusta migratoria (Orthoptera: Acrididae) in Laboratory. Agronomy 2025, 15, 364. https://doi.org/10.3390/agronomy15020364

AMA Style

Zhang P, Yang M, You Y, Zhang L. Synergistic Effect of Mixture of Microsporidium Nosema locustae (Protista: Microsporidia) and Novel Fungus Aspergillus oryzae XJ-1 (Eurotiales: Trichocomaceae) Against Adult Locusta migratoria (Orthoptera: Acrididae) in Laboratory. Agronomy. 2025; 15(2):364. https://doi.org/10.3390/agronomy15020364

Chicago/Turabian Style

Zhang, Pengfei, Mingquan Yang, Yinwei You, and Long Zhang. 2025. "Synergistic Effect of Mixture of Microsporidium Nosema locustae (Protista: Microsporidia) and Novel Fungus Aspergillus oryzae XJ-1 (Eurotiales: Trichocomaceae) Against Adult Locusta migratoria (Orthoptera: Acrididae) in Laboratory" Agronomy 15, no. 2: 364. https://doi.org/10.3390/agronomy15020364

APA Style

Zhang, P., Yang, M., You, Y., & Zhang, L. (2025). Synergistic Effect of Mixture of Microsporidium Nosema locustae (Protista: Microsporidia) and Novel Fungus Aspergillus oryzae XJ-1 (Eurotiales: Trichocomaceae) Against Adult Locusta migratoria (Orthoptera: Acrididae) in Laboratory. Agronomy, 15(2), 364. https://doi.org/10.3390/agronomy15020364

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