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Article

Evaluation of Laboratory Toxicities and Field Application of Plant Spray Oil and Its Mixture with Metarhizium anisopliae Against Diaphorina citri Kuwayama (Hemiptera: Liviidae)

by
Dasong Chen
1,†,
Jiaqi Suo
1,2,†,
Jianquan Yan
1,2,
Lijia Chen
3,
Fenghao Chen
1,2,
Jianying Huang
4,
Haitao Duan
5,
Gecheng Ouyang
1,* and
Xiang Meng
1,*
1
Guangdong Key Laboratory of Animal Conservation and Resource Utilization, Guangdong Public Laboratory of Wild Animal Conservation and Utilization, Institute of Zoology, Guangdong Academy of Science, Guangzhou 510260, China
2
College of Plant Protection, South China Agricultural University, Guangzhou 510642, China
3
College of Life Sciences, Guangzhou University, Guangzhou 510006, China
4
Qingyuan Agricultural Science and Technology Extension Service Center, Qingyuan 511518, China
5
Fogang Chutou Baba Ecological Agriculture Co., Ltd., Qingyuan 511600, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Insects 2025, 16(7), 663; https://doi.org/10.3390/insects16070663
Submission received: 14 March 2025 / Revised: 18 June 2025 / Accepted: 19 June 2025 / Published: 25 June 2025
(This article belongs to the Section Insect Pest and Vector Management)
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Simple Summary

Diaphorina citri, the natural transmission vector of the destructive disease Huanglongbing, is the most important pest in citrus production. This research provides a method for the eco-friendly control of D. citri. A new plant spray oil, based on plant glycerides extracted from soy sauce waste residue, was developed into an environmentally friendly pesticide, which can quickly and effectively control D. citri. When mixed with Metarhizium anisopliae, it had a significant synergistic effect. Field application showed that the control efficacy of the plant spray oil and M. anisopliae mixture on D. citri was equivalent to that of chemical control, presenting a viable alternative for eco-friendly pest management. This study provides a scientific basis for effectively controlling D. citri and preventing the further spread of Huanglongbing in the field.

Abstract

Overusing chemicals to manage Diaphorina citri has created insecticide resistance and negative impacts on the natural ecosystem. This has prompted the need to develop new methods of control. In the present study, we evaluated the toxicity of plant spray oil, Metarhizium anisopliae, and their combined formulations against D. citri adults through laboratory bioassays. We tested varying concentrations and application doses of the individual agents and their mixtures to determine synergistic effects. The optimal mixing ratio of M. anisopliae and plant spray oil for enhanced pest control efficacy was also identified. The results showed that the control effect of plant spray oil and M. anisopliae on D. citri became more pronounced as the concentration increased. When the two were mixed in different proportions, the synergistic effect of plant spray oil was the strongest when the mixing ratio was 5:5. Field trials demonstrated that a combined treatment of plant spray oil emulsion (9.10 g/L) and M. anisopliae (1 × 108 spores/mL) achieved over 80% relative control efficacy against adult D. citri by the 6th day post-treatment. Its efficacy further increased to 96.28% by the 8th day, demonstrating its potential to replace chemical control methods. This study provides a practical example for exploring biopesticides and leveraging the synergistic effects of biogenic pesticides in preventing and controlling pests.

1. Introduction

The Asian citrus psyllid (ACP), Diaphorina citri Kuwayama (Hemiptera: Psyllidae), is a natural vector insect that transmits Huanglongbing (HLB), the most devastating disease of citrus worldwide [1,2,3]. To date, the primary management measure for D. citri has been chemical control by using insecticides [4,5]. It is well-known that excessive insecticide applications may result in negative consequences such as environmental pollution [6], the development of insecticide resistance [7,8], and negative impacts on natural enemy populations [9]. Therefore, to mitigate the adverse effects of insecticides, the use of eco-friendly control agents, such as botanical pesticides and entomopathogenic fungi (EPFs), may be a better strategy for Integrated Pest Management (IPM) [10].
Over more than a century, EPFs have become one of the most critical tools in biological pest control due to their strong pathogenicity, diverse host range, and environmental friendliness [11,12]. Among EPFs, Metarhizium anisopliae (Hypocreales: Clavicipitaceae) is well-known and reported to infect many sucking pests [13,14]. However, the field application of M. anisopliae has received limited attention due to its slower action compared to conventional methods in achieving sufficient insect mortality, and spore germination is adversely affected by ultraviolet radiation under natural conditions [15]. Recently, many studies have explored different strategies to enhance the virulence of these fungi, such as the expression of Bt toxin proteins in Beauveria bassiana, in applications with natural enemies, synthetic oils, and chemical pesticides [16,17].
Plant spray oil is derived from plant glycerol esters extracted from plant sauce waste and formulated into a bio-derived pesticide through specialized processing [18]. Its insecticidal property is mainly contact-based, which forms an oil film over the surface of the insect body and seals off the stomata, egg stomata, or receptors of the insect, leading to asphyxiation. Moreover, plant spray oil also acts as a pest repellent, interfering with critical behaviors such as host positioning, egg laying, and other behaviors [18]. In addition to its insecticidal effects, plant spray oil can enhance the microenvironment for fungal activity, thereby protecting and boosting the pest lethality of fungal spores [19,20]. However, the combined application of plant spray oil with M. anisopliae for the control of D. citri has not yet been explored.
In this study, we investigated the toxicity of plant spray oil, M. anisopliae, and their mixtures against D. citri. The optimal proportion with synergism was screened by a cross-assay and co-toxicity factor method. This study not only provides a technical reference for the green control of D. citri but also provides practical guidance for the field control of D. citri. The experimental results can be further validated in the field.

2. Materials and Methods

2.1. Insects

Adults of D. citri were collected from Murraya paniculata (L.) Jack (Sapindales: Rutaceae) (which is D. citri’s favorite host) at the Sun Yat-sen University (Lat: 23.0966° N, Lng: 113.2986° E), and then they were reared together with a M. paniculata plant on a multi-tier plant cultivation shelf in an artificial isolated climate room with a temperature of 26 ± 1 °C, a relative humidity of 75 ± 5%, and a photoperiod of 14 h:10 h (L:D). Prior to experimentation, the insect colony was maintained for more than three successive generations.

2.2. Plant Spray Oil

The plant spray oil consisted of 93% plant glyceride emulsion, generously provided by Guangzhou European Union Biotechnology Company Ltd. (Guangzhou, China). The plant glyceride emulsion was dissolved with deionized water to prepare a 50-fold dilution solution of plant spray oil emulsion (18.2 g/L; v/v = 1:50). Lower doses of plant spray oil (9.10, 4.55, 2.28, and 1.14 g/L; v/v = 1:100, 1:200, 1:400, 1:800) were obtained through a series dilution method for subsequent experiments.

2.3. Metarhizium anisopliae Culture

Metarhizium anisopliae was isolated from the corpse of D. citri, which was naturally infected, and obtained through strain identification and optimization. The strain was stored at the Environmental Insect Research Center of the Institute of Zoology, Guangdong Academy of Sciences. The strain was cultured in Petri dishes (9 cm dia.) that contained PDA medium and incubated at 26 ± 1 °C for 7 days in darkness. After complete sporulation, spores were collected from the plates.
The fungal conidia were suspended in sterile deionized water containing 0.2% Tween-80 solution. The conidia were counted by using a hematology analyzer under a compound microscope. Then, the conidial suspension was adjusted to 2 × 108, 1 × 108, 5 × 107, and 2.5 × 107 conidia/mL suspension.

2.4. Bioassay

2.4.1. Toxicity of Plant Spray Oil to Diaphorina citri

Two sets of parallel experiments with a high dose of 10 mL and a low dose of 5 mL were conducted on the adults of D. citri using an identical methodology. Fresh M. paniculata tender shoots were selected and placed in a net bag (25 cm × 30 cm), with each bag containing 20 adults of D. citri that were at the same growth period and activity. A hand-held sprayer was used to evenly spray plant spray oil and deionized water at five different concentration gradients onto the surface of the net bags. Following the spraying, the bags were placed in a light incubator set at 26 ± 1 °C with a relative humidity of 75 ± 5% and a photoperiod of 12 L:12 D. We utilized 0.02% Tween-80 sterile water as a control treatment. Adults of D. citri were randomly collected and subjected to three independent biological replications on distinct plants. The number of deaths was recorded at 3 and 6 h after treatment, and the corrected mortality and virulence were calculated.

2.4.2. Toxicity of Metarhizium anisopliae to Diaphorina citri

The spray application method was consistent with that described in Section 2.4.1. Following 10 mL spraying, D. citri adults were placed on fresh M. paniculata tender shoots in a light incubator at 26 ± 1 °C, 75 ± 5% RH, and 12 L:12 D for feeding. We utilized 0.02% Tween-80 sterile water as the blank control. Each treatment was replicated three times, with 20 random healthy adults per replicate on distinct plants. Post-experimental plants were relocated from the trial zone and surface-sterilized via solar exposure in outdoor conditions. Adult mortality was examined at 3 h, 6 h, 1 d, 2 d, and 3 d after treatment.

2.4.3. Toxicity of the Mixed Plant Spray Oil and Metarhizium anisopliae to Adults of Diaphorina citri

According to the LC50 and LT50 values derived from the tests in Section 2.4.1 and Section 2.4.2, appropriate concentrations of plant spray oil and M. anisopliae were prepared. Eleven concentration gradients were set up according to different volume ratios, and the proportions of the mixed solutions were as follows: plant spray oil/M. anisopliae = 10:0, 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, 1:9, and 0:10. A blank control consisting of 0.02% Tween-80 sterile water was also included. Each treatment was replicated three times, with 20 adults in each replicate on independent plants to avoid cross-influence. The plants were removed from the test area after experimentation and exposed to outdoor sunlight for sterilization. The spraying method was the same as that in Section 2.4.1. Following 10 mL spraying, fresh M. paniculata tender shoots were transferred into a light incubator at 26 ± 1 °C, 75 ± 5% RH, and 12 L:12 D for 26 h incubation. Subsequently, the interactive test method and co-toxicity factor analysis were employed for detection and analysis.

2.4.4. Field Control Efficacy of Plant Spray Oil and Metarhizium anisopliae Against Diaphorina citri

The field efficacy trial was conducted targeting three-year-old lemon trees in a lemon orchard spanning 33.33 hectares of Cutou Baba Ecological Agriculture Co., Ltd. in Fogang County, Guangdong Province. The absence of insecticide or fertilizer application prevented any interference with the field trials. The field trial encompassed 4 treatments: A: a control group receiving no intervention; B: 0.5 × 108 conidia/mL M. anisopliae + 4.55 g/L plant spray oil; C: 1 × 108 conidia/mL M. anisopliae + 9.10 g/L plant spray oil; D: orchard conventional chemical treatment. Each treatment was replicated three times, which involved five trees per replication, with spraying twice within 10 days during the shoot growth period. Uniform application was ensured across all treatments utilizing a consistent-frequency sprayer, and orchard management was homogeneously implemented for every treatment group.
A five-point sampling method was employed to investigate the population of D. citri adults per replication. Initial surveys were conducted on the day before treatment, with subsequent assessments on the 2nd, 4th, 6th, and 8th day after treatment. According to the statistical results, the population density, population reduction rate, and relative control efficiency were calculated.

2.5. Data Analysis

The experimental data were statistically analyzed using SPSS 19.0 software. The mortality rate and corrected mortality rate were calculated. The virulence regression equation, LC50, LT50, co-toxicity coefficient, and 95% confidence limits were determined. One-way analysis of variance was used to analyze the experimental data of the decreasing rate of the pest population, relative control efficacy, mortality rate, and corrected mortality rate. And the mean differences between the treatments were tested by ANOVA at a 5% level of significance. Multi-factor analysis of variance was used to determine the influence of plant spray oil, M. anisopliae, and exposure time on the mortality rate of adult D. citri. The calculation formulas are as follows:
The mortality rate was used to calculate the proportion or percentage of pests in a trial that die within a specific time period:
Mortality rate = number of dead insects/number of tested insects × 100%
The corrected mortality was used to calculate the statistical correction to the test mortality rate with the control mortality rate:
Adjusted mortality = (treatment group mortality rate − control group mortality rate)/(1 − control group mortality rate) × 100%
The co-toxicity coefficient (CTF) was used to evaluate the interactive effects (e.g., synergism, antagonism, or additive effects) of two or more combined agents:
Co-toxicity coefficient = (actual mortality − theoretical mortality)/theoretical mortality × 100%
The decreasing rate measured the treatment’s field efficacy in pest populations:
Decreasing rate of pest = (number of treatment group insects before treatment − number of treatment group insects after treatment)/number of treatment group insects before treatment × 100%
Relative control efficacy is a standardized metric used to evaluate the effectiveness of a pest control treatment by comparing changes in pest populations between treated and control groups, while accounting for natural population fluctuations:
Relative control efficacy = (treatment decreasing rate − contral decreasing rate)/(1 − contral decreasing rate) × 100%

3. Results

3.1. Toxicity of Plant Spray Oil to Diaphorina citri

Our results showed that different concentrations and doses of plant spray oil significantly affected the corrected mortality rates (%) of D. citri compared to the control (Table 1). After plant spray oil treatment, the corrected mortality rate of the highest mass concentration was 83.33 ± 6.01% (5 mL)~100 ± 0.00% (10 mL). Toxicity measurements showed that the LC50 of D. citri decreased with the escalating concentrations of plant spray oil and that lethal activity decreased at 6 h compared to 3 h.

3.2. Toxicity of Metarhizium anisopliae to Diaphorina citri

The toxicity of different concentrations of M. anisopliae spore suspensions to D. citri adults showed that the cumulative mortality of D. citri adults escalated in tandem with both the rising M. anisopliae spore concentration and the prolonged duration of spore exposure (Figure 1). Within the consistent treatment duration, a decline in the cumulative mortality of D. citri was observed as the spore concentration of M. anisopliae decreased (Table 2). Optimal insecticidal outcomes were achieved at the top spore concentration (4 × 108 spores/mL). The mortality rate was significantly higher than that in the blank control group on the first day. By the 2nd day, the mortality and corrected mortality of adults were higher than 90% (Table 2). The control efficacy at a spore concentration of 2.5 × 107 spores/mL against adult D. citri was minimal on the first day. However, the insecticidal impact would be progressively amplified over time, culminating in a mortality rate that markedly transcended that of the control group after 2 days.

3.3. Toxicity of the Mixed Plant Spray Oil and Metarhizium anisopliae to Adults of Diaphorina citri

The synergistic toxicity of the mixture of plant spray oil and M. anisopliae in different proportions against the adults of D. citri was evaluated by an interactive test method. The results at the 6th hour after treatment are depicted in Figure 2. When the mixing ratios of plant spray oil and M. anisopliae were 9:1, 1:9, and 7:3, the actual mortality rates of D. citri closely adhered to the theoretical equivalent line, indicating an additive interaction under these ratios. At the mixing ratios of 2:8, 3:7, 4:6, 5:5, 6:4, and 8:2, the actual mortality rate of D. citri surpassed the theoretical equivalent line, indicating that the mixing ratios of the two pesticides had a robust synergistic effect on the control of D. citri; the longer the vertical line is, the stronger the synergy is. That is, the synergy is 2:8, 8:2, 3:7, 4:6, 6:4, and 5:5 from small to large.
The toxicity of the mixtures with different proportions to the adults of D. citri was analyzed by the co-toxicity factor method. The results are shown in Table 3 (after 26 h of treatment). When the mixed proportions of plant spray oil and M. anisopliae were 1:9, 2:8, 3:7, 4:6, 6:4, 7:3, 8:2, and 9:1, the co-toxicity factors were between −20 and 20, indicating that there was an additive effect between the two. When the mixing ratio was 5:5, the co-toxicity factor was 32.5, which is greater than 20, indicating that the two agents have a synergistic effect when mixed according to this ratio. The largest ratio of co-toxic factors was 5:5, which was consistent with the results of the cross-assay.

3.4. Field Control Efficacy for Diaphorina citri

No significant differences in the insect density of D. citri in each experimental area were observed before application. After application, the control efficacy of plant spray oil and M. anisopliae in mitigating D. citri populations was markedly superior to that of the control group, and was comparable to that of the chemical control group (2 d, F = 65.70, p < 0.001; 4 d, F = 332.08, p < 0.001; 6 d, F = 343.83, p < 0.001; 8 d, F = 641.55, p < 0.001). The potency of the mixture amplified in tandem with the increase in the concentration, exerting a sustained suppressive influence on the demographic expansion of D. citri (Figure 3). Further analysis showed that the relative control efficacy and decreasing rate of pests when using the plant spray oil and M. anisopliae mixture significantly transcended those of the control group (8 d relative control efficacy, F = 93.30, p < 0.001; 8 d decreasing rate of pests, F = 322.20, p < 0.001). Among the treatments, the 8-day post-application spray comprising 1 × 108 conidia/mL M. anisopliae + 9.10 g/L plant spray oil yielded the most outstanding control efficacy, reaching 96.28%.

4. Discussion

The employment of eco-friendly and safe pesticides to control plant pests not only safeguards the security of food production but also mitigates pesticide residues and environmental pollution. Moreover, it facilitates the utilization and exploration of the synergism among new green pesticides, thereby offering novel avenues for the prevention and control of field pests. Both plant spray oil and M. anisopliae are widely utilized as green, pollution-free insecticides in agricultural production, but research gaps persist regarding whether their combined application can enhance efficacy against D. citri.
Plant oil derived from fermented foods represents a novel category of plant oils distinguished by its facile environmental degradation, cost-effective production, safety for natural predators, and inability to provoke pest resistance [18]. It plays a pivotal role in the prevention and control of small pests and some foliar microbial diseases. For example, castor oil has been effectively employed to control larvae of Plutella xylostella (Lepidoptera: Plutellidae) in the field, concurrently deterring adults from spawning [21]. The results of our experiments show that plant spray oil can be effectively harnessed against citrus planthopper adults, with its lethal impact on D. citri adults intensifying in tandem with escalating concentrations and dosages of plant spray oil. Consequently, the adoption of high-dose and low-concentration applications of plant spray oil in the field is more in line with the principles of sustainable pest management.
To date, many EPFs have been reported for the control of D. citri, including B. bassiana, M. anisopliae, Aspergillus fijiensis, Paecilomyces variotii, Hirsutella citriformis, and Akanthomyces lecanii [22,23,24,25,26]. Among them, M. anisopliae, which has been commercially produced and applied, is a prominent entomopathogenic fungus widely distributed in nature. It is well-established that M. anisopliae has already been effective against over 200 insect species, encompassing Ceratitis capitata Wiedemann (Diptera: Tephritidae) [27], Spodoptera littoralis (Lepidoptera: Noctuidae) [28], Spodoptera exigua (Lepidoptera: Noctuidae) [29], and Aphis gossypii Glover (Hemiptera: Aphididae) [30], among others. Our results showed that the control effect of M. anisopliae on adults of D. citri was noticeable (89.98%, 92.90%, and 100% mortality rates with 1 × 108, 2 × 108, and 3 × 108 conidia/mL on 3rd day). The efficacy in controlling adults of D. citri is superior to that in previously reported studies, which also found that M. anisopliae isolates caused a severe infection in D. citri (60% with 1 × 108 conidia/mL) [31]. Nevertheless, the common problem faced by our research is that when M. anisopliae was used alone, its effect manifested gradually over a relatively prolonged period, the initial mortality rate was modest, and it had environmental sensitivity [32,33,34].
Plant oil not only exhibits a contact and repellent effect on pests but also can be synergistically combined with other control factors. Plant oil can serve as an adjuvant to facilitate the dispersion, wetting, and deposition of other chemical pesticides on the surface of organisms, thereby enhancing the penetration and coverage of pesticides on the plant surface [18]. This significantly boosts efficiency while decreasing chemical pesticide dosage. In this study, the combination of M. anisopliae and plant spray oil at a 5:5 ratio showed the highest toxicity to adult D. citri. In theory, the mixture ratio of 5:5 can significantly enhance the efficacy while decreasing the dosage requirement of M. anisopliae. Some studies reported that EPFs achieved control at percentages of 35–60% in nymphs and 22–50% in adults [29]. Our trials demonstrate that application comprising 1 × 108 conidia/mL M. anisopliae + 9.10 g/L plant spray oil yielded outstanding control efficacy surpassing 95%. This control efficacy can drive farmers’ willingness to substitute chemical pesticides with fungal insecticides.
Green pest control, an emergent concept put forward in recent years, inclusively leverages diverse methodologies and green pesticides to control the occurrence of diseases and pests. However, practical application has revealed that most green pesticides exhibit modest insecticidal efficacy, which restricts their popularization. Exploring the synergistic interaction among green pesticides and enhancing their insecticidal efficacy are pivotal for facilitating their utilization and popularization. The results of this study play an important guiding role in conforming to the development trend of green agriculture and pollution-free agriculture, giving full play to the synergistic effect of microbe and organic spray oil, and mitigating pesticide usage. Furthermore, plant oil and M. anisopliae are greatly affected by environmental factors such as temperature and humidity, leading to inconsistent control effects and durations in the field. Therefore, the toxicity of the combination of plant spray oil and M. anisopliae to D. citri needs to be verified in additional field trials to support the application of the mixture of plant spray oil and M. anisopliae in the control of D. citri.

Author Contributions

Conceptualization and Methodology, X.M., G.O. and D.C.; Investigation, J.S., L.C., F.C., J.H. and H.D.; Software and Formal analysis, D.C., L.C., F.C., J.H. and H.D. Resources, X.M., G.O., J.H. and H.D. Data curation, J.S., D.C. and J.Y.; Writing—original draft preparation, D.C., J.S., J.Y., L.C. and F.C.; Writing—review and editing, X.M., G.O., J.Y., D.C. and J.S.; Supervision, X.M.; Project administration, G.O. and X.M.; Funding acquisition, G.O. and X.M. All authors have read and agreed to the published version of the manuscript.

Funding

This study was supported by the Science and Technology Program of Guangdong Province (International Cooperation, 2023A0505050142), Science and Technology Projects in Guangzhou (2023B03J1272; 2024B03J1370), Ten Key Projects of Agricultural Science and Technology Innovation in the 14th Five-Year Plan of Guangdong Province (2022SDZG06), and GDAS’ Project of Science and Technology Development (2022GDASZH-2022010106; 2022GDASZH-2022020402-01).

Data Availability Statement

The raw data supporting the conclusions of this article will be made available by the authors on request.

Acknowledgments

The authors are highly thankful to Guangzhou Oulian Biotechnology Co., Ltd. for providing the plant spray oil for this experiment.

Conflicts of Interest

Author Haitao Duan was employed by the Fogang Chutou Baba Ecological Agriculture Co., Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

References

  1. Grafton-Cardwell, E.E.; Stelinski, L.L.; Stansly, P.A. Biology and Management of Asian citrus psyllid, vector of the Huanglongbing pathogens. Annu. Rev. Entomol. 2013, 58, 413–432. [Google Scholar] [CrossRef] [PubMed]
  2. Bové, J.M. Huanglongbing: A destructive, newly-emerging, century-old disease of citrus. Plant Pathol. 2006, 88, 7–37. [Google Scholar]
  3. Gottwald, T.R. Current epidemiological understanding of citrus huanglongbing. Annu. Rev. Phytopathol. 2010, 48, 119–139. [Google Scholar] [CrossRef] [PubMed]
  4. Boina, D.R.; Bloomquist, J.R. Chemical control of the Asian citrus psyllid and of huanglongbing disease in citrus. Pest Manag. Sci. 2015, 71, 808–823. [Google Scholar] [CrossRef]
  5. Miranda, M.P.; dos Santos, F.L.; Bassanezi, R.B.; Montesino, L.H.; Barbosa, J.C.; S’etamou, M. Monitoring methods for Diaphorina citri Kuwayama (Hemiptera: Liviidae) on citrus groves with different insecticide application programmes. J. Appl. Entomol. 2018, 142, 89–96. [Google Scholar] [CrossRef]
  6. Tiwari, S.; Pelz-Stelinski, K.; Stelinski, L.L. Effect of Candidatus Liberibacter asiaticus infection on susceptibility of Asian citrus psyllid, Diaphorina citri, to selected insecticides. Pest Manag. Sci. 2011, 67, 94–99. [Google Scholar] [CrossRef]
  7. Kanga, L.H.B.; Eason, J.; Haseeb, M.; Qureshi, J.; Stansly, P. Monitoring for insecticide resistance in Asian citrus psyllid (Hemiptera: Psyllidae) populations in Florida. Econ. Entomol. 2016, 109, 832–836. [Google Scholar] [CrossRef]
  8. Chen, X.; Stelinski Lukasz, L. Resistance management for Asian citrus psyllid, Diaphorina citri Kuwayama, in Florida. Insects 2017, 8, 103. [Google Scholar] [CrossRef]
  9. Monzo, C.; Qureshi, J.A.; Stansly, P.A. Insecticide sprays, natural enemy assemblages and predation on Asian citrus psyllid, Diaphorina citri (Hemiptera: Psyllidae). Bull. Entomol. Res. 2014, 104, 576–585. [Google Scholar] [CrossRef]
  10. Orduño-Cruz, N.; Guzmán-Franco, A.W.; Rodríguez-Leyva, E.; Alatorre-Rosas, R.; González-Hernández, H.; Mora-Aguilera, G. In vivo selection of entomopathogenic fungal isolates for control of Diaphorina citri (Hemiptera: Liviidae). Biol. Control 2015, 90, 1–5. [Google Scholar] [CrossRef]
  11. Ali, S.; Zhang, C.; Wang, Z.Q.; Wang, X.-M.; Wu, J.-H.; Cuthbertson, A.G.S.; Shao, Z.; Qiu, B.-L. Toxicological and biochemical basis of synergism between the entomopathogenic fungus Lecanicillium muscarium and the insecticide matrine against Bemisia tabaci (Gennadius). Sci. Rep. 2017, 7, 46558. [Google Scholar] [CrossRef] [PubMed]
  12. Islam, W.; Adnan, M.; Shabbir, A.; Naveed, H.; Abubakar, Y.S.; Qasim, M.; Tayyab, M.; Noman, A.; Nisar, M.S.; Khan, K.A.; et al. Insect-fungal-interactions: A detailed review on entomopathogenic fungi pathogenicity to combat insect pests. Microb. Pathog. 2021, 159, 105122. [Google Scholar] [CrossRef] [PubMed]
  13. Naik, P.R.H.; Shekharappa, S. Field evaluation of different entomopathogenic fungal formulations against sucking pests of okra. Karnataka Agric. Sci. 2009, 22, 575–578. [Google Scholar]
  14. Lopes, R.B.; Alves, S.B.; Tamai, M.A. Control of Frankliniella occidentalis in hidroponic lettuce by Metarhizium anisopliae. Sci. Agric. 2000, 57, 239–243. [Google Scholar] [CrossRef]
  15. Kumar, A.; Suroshe, S.S.; Sonam Saini, G.K.; Singh, J. Efficacy of genetically transformed Metarhizium anisopliae against Spodoptera litura and Aphis craccivora. Saudi J. Biol. Sci. 2022, 30, 103493. [Google Scholar] [CrossRef]
  16. Bukhari, T.; Takken, W.; Koenraadt, C.J. Development of Metarhizium anisopliae and Beauveria bassiana formulations for control of malaria mosquito larvae. Parasites Vectors 2011, 4, 23. [Google Scholar] [CrossRef]
  17. Deng, S.Q.; Zou, W.H.; Li, D.L.; Chen, J.-T.; Huang, Q.; Zhou, L.-J.; Tian, X.-X.; Chen, Y.-J.; Peng, H.-J.; Kittayapong, P. Expression of Bacillus thuringiensis toxin Cyt2Ba in the entomopathogenic fungus Beauveria bassiana increases its virulence. PLoS Neglected Trop. Dis. 2019, 13, 1935–2735. [Google Scholar] [CrossRef]
  18. Chen, F.H.; Ouyang, G.C. Research and application of green pesticide-agricultural spray oils. J. Emiron. Entomol. 2024, 46, 43–51. [Google Scholar]
  19. Muniz, E.R.; Paixão, F.R.S.; Barreto, L.P.; Luz, C.; Arruda, W.; Angelo, I.C.; Fernandes, É.K.K. Efficacy of Metarhizium anisopliae conidia in oil-in-water emulsion against the tick Rhipicephalus microplus under heat and dry conditions. BioControl 2000, 65, 339–351. [Google Scholar] [CrossRef]
  20. Batta, Y.A. Invert emulsion: Method of preparation and application as proper formulation of entomopathogenic fungi. MethodsX 2016, 3, 119–127. [Google Scholar] [CrossRef]
  21. Kodjo, T.A.; Gbénonchi, M.; Sadate, A.; Komi, A.; Yaovi, G.; Dieudonné, M.; Komla, S. Bio-insecticidal effects of plant extracts and oil emulsions of Ricinus communis L. (Malpighiales: Euphorbiaceae) on the diamondback, Plutella xylostella L. (Lepidoptera: Plutellidae) under laboratory and semi-field conditions. Appl. Biosci. 2011, 43, 2899–2914. [Google Scholar]
  22. Cortez-Madrigal, H.; Sánchez-Saavedra, J.M.; Díaz-Godínez, G.; Mora-Aguilera, G. Enzymatic activity and pathogenicity of entomopathogenic fungi from central and southeastern Mexico to Diaphorina citri (Hemiptera: Psyllidae). Southwest. Entomol. 2014, 39, 491–502. [Google Scholar] [CrossRef]
  23. Gandarilla-Pacheco, F.L.; Galán-Wong, L.J.; López-Arroyo, J.I.; Rodríguez-Guerra, R.; Quintero-Zapata, I. Optimization of pathogenicity tests for selection of native isolates of entomopathogenic fungi isolated from citrus growing areas of México on adults of Diaphorina citri Kuwayama (Hemiptera: Liviidae). Fla. Entomol. 2013, 96, 187–195. [Google Scholar] [CrossRef]
  24. Hussain, M.; Akutse, K.S.; Lin, Y.; Chen, S.; Huang, W.; Zhang, J.; Idrees, A.; Qiu, D.; Wang, L. Susceptibilities of Candidatus Liberibacter asiaticus-infected and noninfected Diaphorina citri to entomopathogenic fungi and their detoxification enzyme activities under different temperatures. Microbiologyopen 2018, 7, e00607. [Google Scholar] [CrossRef]
  25. Naeem, A.; Freed, S.; Akmal, M. Biochemical analysis and pathogenicity of entomopathogenic fungi to Diaphorina citri Kuwayama (Hemiptera: Liviidae). Entomol. Res. 2020, 50, 245–254. [Google Scholar] [CrossRef]
  26. Yan, J.Q.; Liu, H.; Idrees, A.; Chen, F.H.; Lu, H.L.; Ouyang, G.C.; Meng, X. First Record of Aspergillus fijiensis as an Entomopathogenic Fungus against Asian Citrus Psyllid, Diaphorina citri Kuwayama (Hemiptera: Liviidae). J. Fungi 2022, 8, 1222. [Google Scholar] [CrossRef]
  27. Castillo, M.; Moya, P.; Hernández, E.; Primo-Yufera, E. Susceptibility of Ceratitis capitata Wiedemann (Diptera: Tephritidae) to entomopathogenic fungi and their extracts. Biol. Control 2000, 19, 274–282. [Google Scholar] [CrossRef]
  28. Suarez-Lopez, Y.A.; Aldebis, H.K.; Hatem, A.-S.; Vargas-Osuna, E. Interactions of entomopathogens with insect growth regulators for the control of Spodoptera littoralis (Lepidoptera: Noctuidae). Biol. Control 2022, 170, 104910. [Google Scholar] [CrossRef]
  29. Driver, F.; Milner, R.J.; Trueman, J.W. A taxonomic revision of Metarhizium based on a phylogenetic analysis of rDNA sequence data. Mycol. Res. 2000, 104, 134–150. [Google Scholar] [CrossRef]
  30. Nawaz, A.; Razzaq, F.; Razzaq, A.; Gogi, M.D.; Fernández-Grandon, G.M.; Tayib, M.; Ayub, M.A.; Sufyan, M.; Shahid, M.R.; Qayyum, M.A.; et al. Compatibility and synergistic interactions of fungi, Metarhizium anisopliae, and insecticide combinations against the cotton aphid, Aphis gossypii Glover (Hemiptera: Aphididae). Sci. Rep. 2022, 12, 4843. [Google Scholar] [CrossRef]
  31. Ibarra-Cortés, K.H.; Guzmán-Franco, A.W.; González-Hernández, H.; Ortega-Arenas, L.D.; Villanueva-Jiménez, J.A.; Robles-Bermúdez, A. Susceptibility of Diaphorina citri (Hemiptera: Liviidae) and its parasitoid Tamarixia radiata (Hymenoptera: Eulophidae) to entomopathogenic fungi under laboratory conditions. Neotrop. Entomol. 2018, 47, 131–138. [Google Scholar] [CrossRef] [PubMed]
  32. Han, J.H.; Jin, B.R.; Kim, J.J.; Lee, S.Y. Virulence of entomopathogenic fungi Metarhizium anisopliae and Paecilomyces fumosoroseus for the microbial control of Spodoptera exigua. Mycobiology 2014, 42, 385–390. [Google Scholar] [CrossRef] [PubMed]
  33. Kim, H.M.; Jeong, S.G.; Choi, I.S.; Yang, J.E.; Lee, K.H.; Kim, J.; Kim, J.C.; Kim, J.S.; Park, H.W. Mechanisms of Insecticidal Action of Metarhizium anisopliae on adult Japanese pine sawyer beetles (Monochamus alternatus). ACS Omega 2020, 5, 25312–25318. [Google Scholar] [CrossRef] [PubMed]
  34. Lezama-Gutiérrez, R.; Molina-Ochoa, J.; Chávez-Flores, O.; Ángel-Sahagún, C.A.; Skoda, S.R.; Reyes-Martínez, G.; Barba-Reynoso, M.; Rebolledo-Domínguez, O.; Ruíz-Aguilar, G.M.; Foster, J.E. Use of the entomopathogenic fungi Metarhizium anisopliae, Cordyceps bassiana and Isaria fumosorosea to control Diaphorina citri (Hemiptera: Psyllidae) in Persian lime under field conditions. Int. J. Trop. Insect Sci. 2012, 32, 39–44. [Google Scholar] [CrossRef]
Insects 16 00663 g001
Figure 1. Comparison of time–mortality relationship for M. anisopliae used on D. citri adults inoculated with spores at varied concentrations. Spore suspensions were prepared in 0.02% Tween-80 solution at concentrations of 2.5 × 107, 5 × 107, 1 × 108, 2 × 108, and 3 × 108 spores/mL, with the 0.02% Tween-80 solution as the control.
Figure 1. Comparison of time–mortality relationship for M. anisopliae used on D. citri adults inoculated with spores at varied concentrations. Spore suspensions were prepared in 0.02% Tween-80 solution at concentrations of 2.5 × 107, 5 × 107, 1 × 108, 2 × 108, and 3 × 108 spores/mL, with the 0.02% Tween-80 solution as the control.
Insects 16 00663 g001
Insects 16 00663 g002
Figure 2. Synergistic effect of plant spray oil and M. anisopliae on adults of D. citri according to interactive determination method.
Figure 2. Synergistic effect of plant spray oil and M. anisopliae on adults of D. citri according to interactive determination method.
Insects 16 00663 g002
Insects 16 00663 g003
Figure 3. Field control efficacy of D. citri with different treatments in the field. The experimental data were analyzed using one-way ANOVA, and Duncan’s new multiple range test was employed to test the differences among treatments. Different letters indicate significant differences (p < 0.05). Error bars represent the standard error of the mean.
Figure 3. Field control efficacy of D. citri with different treatments in the field. The experimental data were analyzed using one-way ANOVA, and Duncan’s new multiple range test was employed to test the differences among treatments. Different letters indicate significant differences (p < 0.05). Error bars represent the standard error of the mean.
Insects 16 00663 g003
Table 1. Toxicity of different doses of plant spray oil to D. citri adults.
Table 1. Toxicity of different doses of plant spray oil to D. citri adults.
DoseMass Concentration g/LCorrected Mortality/%Regression EquationLC50 (g/L)95% Confidence Intervalχ2
5 mL
(6 h)		1.146.67 ± 4.41 dY = 0.138X − 1.1828.5616.526–11.1584.699
2.2810.00 ± 0.00 d
4.5530.00 ± 5.00 c
9.1065.00 ± 0.00 b
18.283.33 ± 6.01 a
10 mL
(3 h)		1.1413.56 ± 2.94 eY = 0.264X − 1.4915.6384.448–7.2920.996
2.2822.03 ± 3.40 d
4.5532.20 ± 3.40 c
9.1088.14 ± 1.69 b
18.2100.00 ± 0.00 a
Data are mean ± SE. Data from same experiment followed by different letters indicate significant difference at 5% level by Duncan’s new multiple range method. Statistical analyses were performed on each condition separately.
Table 2. Toxicity of different concentrations of M. anisopliae to D. citri adults.
Table 2. Toxicity of different concentrations of M. anisopliae to D. citri adults.
Concentrations
(×107 conidia/mL)		Corrected Mortality/%
(1 d)		LC50
(×107 conidia/mL)		Corrected Mortality/%
(3 d)		LT50/hRegression
Equation		95% Confidence Intervalχ2
4088.48 ± 2.15 a10.082100 ± 0 a10.660Y = 0.063X − 0.5504.854–16.2662.227
2064.36 ± 5.41 b92.90 ± 3.49 ab21.453Y = 0.036X − 0.77713.331–29.0362.453
1059.52 ± 3.37 b89.98 ± 7.09 b26.070Y = 0.035X − 0.92117.737–34.3843.186
547.50 ± 3.56 c87.56 ± 2.70 b32.342Y = 0.035X − 1.13824.289–41.1711.750
2.524.41 ± 0.84 d84.59 ± 0.53 b39.209Y = 0.039X − 1.52731.606–48.1331.88
Data are mean ± SE. Data from same experiment followed by different letters indicate significant difference at 5% level by Duncan’s new multiple range method.
Table 3. Synergistic effect of plant spray oil and M. anisopliae on adults of D. citri according to co-toxicity factor (CTF) method.
Table 3. Synergistic effect of plant spray oil and M. anisopliae on adults of D. citri according to co-toxicity factor (CTF) method.
Plant Spray Oil/M. anisopliaeActual MortalityTheoretical MortalityCTF
0:1048.14848.1480
1:950.00053.333−6.249
2:866.66758.51813.926
3:775.92663.70319.188
4:681.48168.88918.279
5:598.14874.07432.500
6:492.59379.25916.823
7:381.48184.444−3.509
8:2100.00089.63011.570
9:198.14894.8153.515
10:0100.000100.0000
Note: CTF: co-toxicity coefficient; CTF > 20: synergistic interaction; CTF < −20: antagonistic interaction; −20 ≤ CTF ≤ 20: additive interaction.
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Chen, D.; Suo, J.; Yan, J.; Chen, L.; Chen, F.; Huang, J.; Duan, H.; Ouyang, G.; Meng, X. Evaluation of Laboratory Toxicities and Field Application of Plant Spray Oil and Its Mixture with Metarhizium anisopliae Against Diaphorina citri Kuwayama (Hemiptera: Liviidae). Insects 2025, 16, 663. https://doi.org/10.3390/insects16070663

AMA Style

Chen D, Suo J, Yan J, Chen L, Chen F, Huang J, Duan H, Ouyang G, Meng X. Evaluation of Laboratory Toxicities and Field Application of Plant Spray Oil and Its Mixture with Metarhizium anisopliae Against Diaphorina citri Kuwayama (Hemiptera: Liviidae). Insects. 2025; 16(7):663. https://doi.org/10.3390/insects16070663

Chicago/Turabian Style

Chen, Dasong, Jiaqi Suo, Jianquan Yan, Lijia Chen, Fenghao Chen, Jianying Huang, Haitao Duan, Gecheng Ouyang, and Xiang Meng. 2025. "Evaluation of Laboratory Toxicities and Field Application of Plant Spray Oil and Its Mixture with Metarhizium anisopliae Against Diaphorina citri Kuwayama (Hemiptera: Liviidae)" Insects 16, no. 7: 663. https://doi.org/10.3390/insects16070663

APA Style

Chen, D., Suo, J., Yan, J., Chen, L., Chen, F., Huang, J., Duan, H., Ouyang, G., & Meng, X. (2025). Evaluation of Laboratory Toxicities and Field Application of Plant Spray Oil and Its Mixture with Metarhizium anisopliae Against Diaphorina citri Kuwayama (Hemiptera: Liviidae). Insects, 16(7), 663. https://doi.org/10.3390/insects16070663

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