AgBioForum, 26(3): 148-155. ©2024 AgBioForum Xuan et al — Biocontrol Potential of Different Entomopathogenic Fungi and Neem Oil for Control of Sucking Insects and Two-Spotted... Biocontrol Potential of Different Entomopathogenic Fungi and Neem Oil for Control of Sucking Insects and Two-Spotted Spider Mites in Vegetables Trinh Thi Xuan* Plant Protection Department, College of Agriculture, Can Tho University, Can Tho City, Vietnam. ORCID iD: https://orcid.org/0009-0002-0513-79222 Email: trinhthixuan@ctu.edu.vn Cao Thi Tuyet Mai An Giang University, Long Xuyen City, An Giang, Vietnam. ORCID iD: https://orcid.org/0009-0008-5434-6302 Email: caothituyetmai2010@gmail.com Nguyen Hong Ung Tra Vinh University, Tra Vinh, Vietnam. ORCID iD: https://orcid.org/0009-0003-2488-0583 Email: nghongung@tvu.edu.vn Lam Thi Xuan Mai Plant Protection Department, College of Agriculture, Can Tho University, Can Tho City, Vietnam. ORCID iD: https://orcid.org/0009-0007-5973-469X Email: ltxmai@ctu.edu.vn Pham Van Hiep Dong Thap University, Cao Lanh, Dong Thap, Vietnam. ORCID iD: https://orcid.org/0009-0004-2934-8964 Email: pvhiep@dthu.edu.vn Nguyen Thi Kieu Bac Lieu University, Bac Lieu, Vietnam.x ORCID iD: https://orcid.org/0009-0008-5365-9331 Email: ntkieu@blu.edu.vn Pham Kim Son Plant Protection Department, College of Agriculture, Can Tho University, Can Tho City, Vietnam. ORCID iD: https://orcid.org/0009-0004-9194-5719 Email: pkson@ctu.edu.vn Greenhouse vegetable crops are significantly affected by infestations from three major groups of harmful sucking insects: aphids (Aphis gossypii), whiteflies (Bemisia tabaci), and two- spotted spider mites (Tetranychus urticae). Aim: The study aimed to assess the effectiveness of entomopathogenic fungi, including Metarhizium anisopliae, Beauveria bassiana, and Paecilomyces fumosoroseus, both individually and in combination with neem oil, against these pests. When treatments were applied six days prior to testing, all entomopathogens at a concentration of 100,000 spores per milliliter resulted in 84% mortality of T. urticae, A. gossypii, and B. tabaci. Higher doses of neem oil, when combined with entomopathogenic fungi, proved effective in controlling insect pests. The combination of entomopathogenic fungi at a concentration of 1 x 10⁵ spores/mL and 1% neem oil in a 1:1 ratio exhibited significantly greater pest elimination success compared to the individual treatments. Specifically, the combination of M. anisopliae and neem oil (1:1) achieved 100% mortality of T. urticae and A. gossypii, while P. fumosoroseus with neem oil (1:1) was effective against B. tabaci six days post-treatment. This research demonstrated that combining entomopathogenic fungi with neem oil in a 1:1 ratio proved more effective than applying them individually. The study also highlighted the compatibility of these treatments, with all experimental samples resulting in mortality rates exceeding 100%. Keywords: Biocontrol, Entomopathogenic Fungi, Neem Oil, Sucking Insects, Two-Spotted Spider Mite. Introduction In recent times, modifications in farming techniques, the progression of climate change, and the increased utilisation of input-intensive, high-yield crop varieties have significantly altered pest dynamics in the Mekong Delta region of Vietnam. These shifts have notably impacted vegetable ecosystems and orchards. Changes in both environmental and ecological parameters have contributed to the emergence of several insect pests and mites, which previously posed minimal threats to vegetable production in the region, as pressing concerns across various localities in the country (Deguine et al., 2023; Van Mele et al., 20010). Among these, sucking insect pests and two- spotted spider mites, specifically Aphis gossypii, Bemisia tabaci, and Tetranychus urticae, have gained recognition as critical constraints to vegetable cultivation not only in Vietnam but also globally (Van Mele, Cuc, & Van Huis, 2010; Van Mele et al., 20010). These pests are known for their polyphagous nature and feed on a wide spectrum of host plants such as cabbage, tomatoes, eggplants, and ornamental species like roses and lilies. Their geographical distribution spans extensively worldwide, with higher population densities typically recorded in temperate climates, which also exhibit greater species diversity than tropical zones (Żyła, Homan, & Wegierek, 2017). In response to escalating pest infestations, farmers have resorted to frequent and heavy application of insecticides. This approach has led to several adverse outcomes, including the resurgence of targeted pests, the development of insecticide resistance, and the outbreak of secondary pests. Additionally, the residual presence of chemical pesticides in agricultural produce, water sources, and beverages has given rise to severe health and environmental repercussions. These include contamination of groundwater, human health hazards, and the mortality of non-target organisms such as pollinators and beneficial insects (Ambethgar, 2009; Deguine et al., 2023; Halder, Divekar, & Rani, 2021; Van der Sluijs et al., 2013). In this context, biopesticides offer a sustainable alternative by minimising dependency on synthetic chemicals (Deguine et al., 2023). Entomopathogenic fungi (EPFs) have demonstrated considerable efficacy in reducing pest populations across a diverse range of agricultural systems. Species such as Metarhizium anisopliae, Beauveria bassiana, and https://orcid.org/0009-0002-0513-79222 mailto:trinhthixuan@ctu.edu.vn https://orcid.org/0009-0008-5434-6302 mailto:caothituyetmai2010@gmail.com https://orcid.org/0009-0003-2488-0583 mailto:nghongung@tvu.edu.vn https://orcid.org/0009-0007-5973-469X mailto:ltxmai@ctu.edu.vn https://orcid.org/0009-0004-2934-8964 mailto:pvhiep@dthu.edu.vn https://orcid.org/0009-0008-5365-9331 mailto:ntkieu@blu.edu.vn https://orcid.org/0009-0004-9194-5719 mailto:pkson@ctu.edu.vn AgBioForum, 26(3), 2024 | 149 Xuan et al — Biocontrol Potential of Different Entomopathogenic Fungi and Neem Oil for Control of Sucking Insects and Two-Spotted... Paecilomyces fumosoroseus are well-documented for their pest control potential. Studies focusing on the biological control of insect pests using EPFs have reported high levels of effectiveness, targeting up to 150 insect species. The most widely utilised biocontrol strains originate from four primary Hypocrealean fungal species: M. anisopliae, B. bassiana, Isaria fumosorosea, and B. brongniartii. Initial commercial trials have likely enhanced these success rates. However, a major limitation of EPFs lies in their relatively slow mode of action, as the time required to achieve insect mortality surpasses that of conventional chemical agents. This drawback can be mitigated by integrating EPFs with faster-acting compounds in pest management systems (Bitsadze et al., 2013). The fungal pathogen M. anisopliae has shown effectiveness against more than 200 insect pest species. Research by González-Mas et al. (2019) identified significant control outcomes for A. gossypii, two-spotted spider mites (TSSM), and other related mite species. Further evidence from studies such as Cakmak et al. (2009) confirms that, following cuticular penetration, EPF conidia rapidly colonise the insect haemocoel, subsequently proliferating and sporulating within the host as well as on its external surface (Chandler, Davidson, & Jacobson, 2005). The success of EPFs in controlling TSSM and other sucking insects is influenced by the fungal strain, the concentration of the inoculum, and prevailing environmental conditions. Azadirachta indica A. Juss (commonly known as neem) has gained widespread application in pest control due to its efficacy against multiple insect species, accessibility, and cost-effective production. Efforts have been made to employ biocontrol agents and botanicals in equal proportions to assess their compatibility and maximise pest control outcomes (Gatarayiha, Laing, & Miller, 2010). The co-application of fungal biopesticides and neem oil has emerged as a promising strategy (Ullah & Lim, 2015). This integrated approach enhances both economic viability and environmental sustainability compared to chemical insecticides (Bugeme et al., 2014). Moreover, there are no recorded cases of resistance development to these biological control methods, and they are considered environmentally safe. Accordingly, the present study aimed to investigate the effectiveness of selected entomopathogenic fungi, neem oil, and their low-concentration combinations in suppressing A. gossypii, B. tabaci, and T. urticae, as part of a biologically based pest management strategy. Materials and Methods Insect Collection and Rearing of A. gossypii, B. tabaci, and T. urticae The present study involved the collection of sucking insect species from eggplant and tomato fields that were free from pest infestation and insecticide exposure in Can Tho City, Vietnam. The identified pests included aphids (Aphis gossypii), whiteflies (Bemisia tabaci species), and two- spotted spider mites (Tetranychus urticae). A research plot maintained by the University was utilised for this purpose. Seedlings were grown under regulated conditions in plastic pots with a diameter of 15 cm, using a sandy loam and organic matter mixture at a 2:1 ratio. The growth environment was maintained at a temperature of 25 ± 1°C with a relative humidity of 65 ± 10%. The pots were enclosed to facilitate the natural reproduction of aphids, whiteflies, and spider mites along with their respective nymphs, ensuring the absence of insecticidal interference. For the bioassay trials, second and third instar nymphs derived from the offspring of the aforementioned insect species were employed. Preparation of Concentration of Entomopathogenic Fungi and Neem Oil The fungal isolates Beauveria bassiana CTUBb28, Metarhizium anisopliae CTUMa01, and Paecilomyces fumosoroseus CTUPae025 were cultured on SDAY3 medium (Sabouraud Dextrose Agar Yeast), which comprised 40 g of dextrose, 10 g of peptone, 10 g of yeast extract, 20 g of agar, 0.5 g of magnesium sulphate (MgSO₄), 1.0 g of monopotassium phosphate (KH₂PO₄), and 1,000 mL of distilled water. The cultures were incubated at a constant temperature of 25°C for a duration of 14 days. Upon completion of the sporulation phase, conidia were collected using an inoculation loop, washed with sterile distilled water, and filtered through a double layer of gauze to remove any residual hyphal structures. To attain the desired concentrations for experimental application, conidial suspensions were prepared in a 0.01% Tween 80 solution. In accordance with the germination assessment procedure outlined by Ansari & Butt (2011), conidial viability was confirmed to exceed 90% across all evaluations. Neem oil emulsions were formulated by blending the oil with an emulsifying agent based on Triton- X-100. Bioassay Bioassays for aphids and whiteflies were carried out, while assessments involving TSSM were performed using Petri dish setups. In these experiments, leaves from eggplant plants were arranged with the abaxial (underside) surface facing upwards, resting on a bed of moistened cotton wool. The petioles of the leaves were inserted into the cotton layer to preserve turgidity and sustain adequate moisture levels throughout the testing period. Effect of Different Doses of Neem Oil Against Aphids, Whiteflies, and TSSM Five different concentrations of neem oil (1%, 2%, 3%, 4%, and 5%) were prepared, with each concentration being replicated three times. A total of 90 nymphs were assigned to each treatment group and placed in separate Petri dishes, each 9 cm in diameter. The mortality rates of aphids, whiteflies, and TSSM were observed individually at 24- hour intervals over a six-day period. Bioassay of Entomopathogenic Against Aphids, Whiteflies, and TSSM The fungal strains Beauveria bassiana, Metarhizium anisopliae, and Paecilomyces fumosoroseus were prepared at four different concentrations: 1 × 10⁴, 1 × 10⁵, 1 × 10⁶, and 1 × 10⁷ conidia/mL. Each concentration was tested in AgBioForum, 26(3), 2024 | 150 Xuan et al — Biocontrol Potential of Different Entomopathogenic Fungi and Neem Oil for Control of Sucking Insects and Two-Spotted... triplicate. For each treatment, 75 nymphs were placed into individual Petri dishes, each with a diameter of 9 cm. The mortality of aphids, whiteflies, and two-spotted spider mites was assessed separately at 24-hour intervals over a six-day period. Bioassay of Neem Oil and Mix with Entomopathogenic Against Aphids, Whiteflies, and TSSM A total of thirty insect samples were placed individually into separate Petri dishes, each with a diameter of 9 cm. Three independent trials were conducted for each experimental condition. The seven experimental groups were as follows: A1: neem oil alone; A2: M. anisopliae; A3: B. bassiana; A4: P. fumosoroseus; A5: M. anisopliae combined with neem oil (1:1); A6: B. bassiana combined with neem oil (1:1); A7: P. fumosoroseus combined with neem oil (1:1); and a control group treated with water. The application method involved the use of Potter's tower, applying different concentrations of neem oil mixed with entomopathogenic fungi and a 1:1 ratio of neem oil for 10 seconds at a rate of 4,000 g/cm². After spraying, the pots containing the treated insects were dried using a fan. The bioassays were performed under controlled laboratory conditions, with temperatures maintained at 25 ± 1°C and a relative humidity of 70 ± 5%. Data Analysis Daily observations were conducted to record the number of deaths among aphids, whiteflies, and TSSM. Abbott’s formula was employed to adjust for control group mortality. The data were subsequently analysed using probit analysis to calculate the median lethal concentration (LC50) and median lethal time (LT50), with statistical computations carried out using the Statistical Package for the Social Sciences. Additionally, the cumulative corrected mortality percentages for each fungal treatment were subjected to analysis of variance (ANOVA), and treatment means were compared using Duncan’s Multiple Range Test (DMRT). C (%) = 𝐴−𝐵 𝐴 × 100 Where: C = Corrected mortality; A = Average number of pests (aphid, whitefly, and TSSM) in the control group; B = Average number of pests (aphid, whitefly, and TSSM) in the treatment group. Results and Discussion Effect of Range Dose of Neem Oil Against Two Kinds of Sucking Insects and TSSM The maximum biological efficacy under laboratory conditions was recorded daily following treatment. The results indicated that, after six days, the treatment with 5% neem oil exhibited the highest efficacy against both sucking insects and two-spotted spider mites Figure. 1. The toxicological effects of varying doses of neem oil and their interactions with the nymphs of T. urticae, A. gossypii, and B. tabaci are illustrated in Figure. 1. For T. urticae, the highest nymph mortality rate (74.3%) was recorded at a 5% dose, though this was not statistically significant when compared to the 4% neem oil dose (62.7%). In contrast, the lowest mortality (45.7%) occurred with a 1% neem oil dose Figure. 1. Nymphs of A. gossypii displayed high susceptibility to neem oil at all concentrations, with mortality rates of 93.3%, 96.7%, and 100% observed at 3%, 4%, and 5%, respectively. Furthermore, the mortality rate of B. tabaci was found to be 90.3% at the 5% dose, which was statistically distinct from the other doses. Figure 1: Corrected Mortality in T. urticae, A. gossypii, and B. tabaci after 6 Days of Application of Neem Oil at Different Doses under Laboratory Conditions. Percent Mortality of Sucking Insects and TSSM to Entomopathogenic Fungi * T. urticae Table 1 illustrates that the mortality percentage of nymphs was 60.0%, 67.6%, and 68.6% after the fifth day for M. anisopliae, B. bassiana, and P. fumosoroseus, respectively, at a concentration of 1 × 10⁷ spores/mL. By the sixth day of infection, the mortality rates for M. anisopliae, B. bassiana, and P. fumosoroseus ranged from 66.7% to 84.8%, 56.2% to 85.7%, and 60.0% to 85.7%, respectively. The results indicated that all entomopathogenic fungi at the 1 × 10⁷ spores/mL concentration were associated with a mortality rate exceeding 84% on the sixth day post-treatment. AgBioForum, 26(3), 2024 | 151 Xuan et al — Biocontrol Potential of Different Entomopathogenic Fungi and Neem Oil for Control of Sucking Insects and Two-Spotted... * A. gossypii Table 1 shows that the highest nymph mortality was recorded with P. fumosoroseus at a concentration of 1 × 10⁷ spores/mL, resulting in 78.3% mortality, followed by M. anisopliae at 68.3%, and B. bassiana at 66.7% at the same concentration. By the sixth day of treatment, fungal applications at 1 × 10⁷ spores/mL reached peak mortality rates, ranging from 85.0% to 91.7% under controlled laboratory conditions. * B. tabaci According to Table 1, M. anisopliae at a concentration of 1 × 10⁷ spores/mL resulted in 50.0% nymph mortality by the fifth day post-infection. At the same concentration, B. bassiana caused 53.3% mortality, while P. fumosoroseus achieved 71.7%. By the sixth day, mortality rates had increased, with M. anisopliae reaching 88.3%, and P. fumosoroseus ranging from 91.7% to 93.3% at concentrations of 1 × 10⁶ and 1 × 10⁷ spores/mL, respectively. Table 1: Percentage Mortality of Nymphs of Sucking Pests and Two-Spotted Spider Mites (TSSM) at Various Time Intervals Following Exposure to Entomopathogenic Fungi (EPF) under Controlled Conditions of 25 ± 1°C and 70 ± 5% Relative Humidity. Treatments Doses (Spores/mL) Corrected Mortality (%) T. urticae A. gossypii B. tabaci 4th DAT 5th DAT 6th DAT 4th DAT 5th DAT 6th DAT 4th DAT 5th DAT 6th DAT M. anisopliae D1 27.6 48.6 66.7 35.0 51.7 76.7 13.3 28.3 70.0 D2 35.2 50.5 78.1 40.0 55.0 85.0 16.7 41.7 78.3 D3 39.1 52.4 82.9 46.7 61.7 85.0 23.3 51.7 91.7 D4 44.8 60.0 84.8 50.0 68.3 88.3 26.7 61.7 93.3 B. bassiana D1 29.5 38.1 56.2 25.0 48.3 68.3 11.7 18.3 70.0 D2 32.4 49.5 68.6 28.3 53.3 78.3 13.3 23.3 73.3 D3 41.9 61.0 76.2 31.7 60.0 81.7 15.0 41.7 86.7 D4 49.5 67.6 85.7 33.3 66.7 85.0 16.7 53.3 81.7 P. fumosoroseus D1 28.6 43.8 60.0 36.7 58.3 81.7 13.3 36.7 80.0 D2 32.4 44.8 61.9 41.7 66.7 83.3 28.3 53.3 81.7 D3 38.1 48.6 68.6 50.0 71.7 90.0 33.3 58.3 83.3 D4 52.4 68.6 85.7 53.3 78.3 91.7 35.0 71.7 88.3 D1: 1 × 10⁴ Spores/mL; D2: 1 × 10⁵ Spores/mL; D3: 1 × 10⁶ Spores/mL; D4: 1 × 10⁷ Spores/mL; DAT: Days after Treatment. The Median Lethal Concentration (LC50) The LC50 values and toxicity levels of the three species of entomopathogenic fungi were compared against sucking insects and TSSM, as shown in Table 2. The results revealed that the lowest LC50 values were 0.87 × 10⁵ spores/mL and 0.10 × 10⁷ spores/mL for M. anisopliae against T. urticae and B. tabaci, respectively. Meanwhile, the lowest LC50 value for P. fumosoroseus was 0.58 × 10³ spores/mL against A. gossypii. Table 2: Dose Mortality of EPF Against Sucking Insects and TSSM 25 ± 1°C and 70 ± 5% R.H. EPFs Dose Mortality of Entomopathogenic Fungi T. urticae A. gossypii B. tabaci LC50 (Spores/mL) 95% Fiducial Limits (Spores/mL) LC50 (Spores/mL) 95% Fiducial Limits (Spores/mL) LC50 (Spores/mL) 95% Fiducial Limits (Spores/mL) M. anisopliae 0.87 x 105 0.21 x 103 - 0.93 x 106 0.81 x 104 0.39 x 101 - 0.12 x 106 0.10 x 107 0.79 x 106 - 0.54 x 109 B. assiana 0.13 x 106 0.28 x 105- 0.38x106 0.17 x 105 0.11 x 102 - 0.20 x 106 0.15 x 108 0.43 x 107 - 0.72 x 108 P. fumosoroseus 0.19 x 106 0.72 x 104- 0.12x107 0.58 x 103 0.27 x 102 - 0.12 x 105 0.60 x 107 0.15 x 107 - 0.28 x 108 The Median Lethal Time (LT50) As shown in Table 3, the LT₅₀ values decreased as the spore concentration increased. At the highest tested concentration of 1 × 10⁷ spores/mL, the LT₅₀ for M. anisopliae, B. bassiana, and P. fumosoroseus was recorded as 3.55, 3.75, and 3.37 days, respectively, for T. urticae. For A. gossypii, the corresponding LT₅₀ values were 2.70, 2.72, and 2.42 days, while for B. tabaci, the LT₅₀ values were 4.04, 4.88, and 4.05 days, respectively. Table 3: Time Mortality of EPF Against Sucking Insects and TSSM 25 ± 1°C and 70 ± 5% R.H. EPFs Median Lethal Time (Days) of Entomopathogenic Fungi T. Urticae A. gossypii B. Tabaci D1 D2 D3 D4 D1 D2 D3 D4 D1 D2 D3 D4 M. anisopliae 4.44 4.04 3.80 3.55 3.32 3.10 2.88 2.70 5.64 5.04 4.30 4.04 B. bassiana 4.80 4.40 3.90 3.75 3.32 3.07 2.85 2.72 5.92 5.80 5.05 4.88 P. fumosoroseus 4.71 4.52 4.15 3.37 3.17 2.85 2.58 2.42 5.41 4.92 4.65 4.05 D1: 1 x 104 spores/mL; D2: 1 x 105 spores/mL; D3: 1 x 106 spores/mL; D4: 1 x 107 spores/mL AgBioForum, 26(3), 2024 | 152 Xuan et al — Biocontrol Potential of Different Entomopathogenic Fungi and Neem Oil for Control of Sucking Insects and Two-Spotted... The Combination of Entomopathogens with Neem Oil Against Sucking Insects and TSSM A. gossypii and B. tabaci The examination of the effects of various entomopathogens, both individually and in combination with neem oil, revealed significant differences among the three types of EPFs, whether used alone or together. M. anisopliae demonstrated substantial potential as an entomopathogen against two sucking insects, aphids and whiteflies. The highest mortality rate observed was 81.1% at a concentration of 10⁵ spores/mL and 75.0% six days after treatment (DAT) on A. gossypii and B. tabaci, respectively. B. bassiana showed corresponding mortality rates of 74.4% and 80.0%, respectively Table 4. In contrast, P. fumosoroseus exhibited the most efficacy against the nymphs of A. gossypii (66.7%, 81.1%, and 87.8% at 4, 5, and 6 DAT, respectively) and B. tabaci (51.24%, 72.88%, and 85.38% at 4, 5, and 6 DAT, respectively). Neem oil (1%), however, showed lower efficacy as a botanical treatment for the emerging pests, with mortality rates at 4, 5, and 6 DAT of 42.2%, 55.6%, and 72.0% for A. gossypii, and 20.0%, 31.67%, and 51.67% for B. tabaci. When these entomopathogenic fungi were applied in combination with neem oil at a 1:1 ratio, mortality rates significantly increased in all cases compared to their individual effects. This suggests compatibility among these essential biopesticides. The treatments combining M. anisopliae and neem oil, as well as P. fumosoroseus and neem oil at a 1:1 ratio, achieved maximum effective control of 100% against A. gossypii Table 4. The mixture of B. bassiana with neem oil resulted in 95.6% pest control. A similar trend was observed in B. tabaci, with complete (100%) control of thrips nymphs attained through the combined application of P. fumosoroseus and neem oil, M. anisopliae with neem oil, and B. bassiana with neem oil. T. urticae The present study highlighted the effectiveness of EPFs and neem oil in controlling TSSM. M. anisopliae exhibited an average effectiveness of 78.1% when used alone, but when combined with neem oil at a 1:1 ratio, it resulted in a maximum mortality rate of 100%. This was followed by a 98.1% efficacy for the combination of P. fumosoroseus and neem oil at the same ratio. The mixture of B. bassiana and neem oil demonstrated an efficacy of 86.7%. Table 4: Impact of Various Entomopathogens Individually and in Combination with Neem Oil on T. urticae, A. gossypii, and B. tabaci at 25 ± 1°C and 70 ± 5% R.H. Treatments The Mortality (%) T. urticae A. gossypii B. tabaci 4th DAT 5th DAT 6th DAT 4th DAT 5th DAT 6th DAT 4th DAT 5th DAT 6th DAT A1 41.9e 48.6e 53.3e 42.2e 55.6f 72.0f 20.00d 31.67d 51.67c A2 65.7c 76.2c 78.1c 61.1cd 73.3de 81.1d 42.5c 61.67c 75.00b A3 51.4de 60.0de 65.7d 55.6d 67.8ef 74.4e 40.00c 65.00c 80.00b A4 58.1cd 68.6cd 74.3cd 66.7c 81.1cd 87.8c 51.24d 72.88b 85.38b A5 92.4a 95.2a 100a 90.0a 95.6ab 100a 49.17c 63.33c 78.33b A6 77.1b 81.0bc 86.7b 84.4b 90.0bc 95.6b 59.61cd 77.62b 88.97b A7 87.6a 92.4a 98.1a 93.3a 97.8a 100a 87.49b 95.47a 100.00a Note: Identical letters within the same column indicate no significant differences (P ≤ 0.05). In this case, A1 refers to neem oil applied at a 1% concentration; A2 corresponds to M. anisopliae at 1 × 10⁵ spores/mL; A3 represents B. bassiana at 1 × 10⁵ spores/mL; and A4 indicates P. fumosoroseus at 1 × 10⁵ spores/mL. Treatments A5, A6, and A7 represent combinations of M. anisopliae, B. bassiana, and P. fumosoroseus with neem oil, respectively, each mixed in a 1:1 ratio. The term DAT denotes the number of days after treatment application. Discussion The potential of entomopathogenic fungi, including Metarhizium anisopliae, Beauveria bassiana, Paecilomyces fumosoroseus, and neem oil, as biological control agents against Tetranychus urticae, Aphis gossypii, and Bemisia tabaci appears promising for future applications. Higher concentrations are likely to enhance efficacy in controlling these insects. The study also highlighted that increased doses of neem oil or fungal conidia concentrations can significantly improve the effectiveness against the targeted pests. When exposed to the same concentration of conidia, Aphis gossypii and Tetranychus urticae showed greater susceptibility to Metarhizium anisopliae CTUMa01 and Paecilomyces fumosoroseus CTUPae025 compared to Beauveria bassiana CTUBb28. Abdel-Raheem, Saad, & Abdel- Rahman (2021) examined the mortality rates of Myzus persicae, Aphis gossypii, and Aphis citricola at spore concentrations between 10⁶ and 10⁷ spores/mL over four days of testing. Saranya et al. (2010) found that Verticillium lecanii and Hirsutella thompsonii achieved 100% mortality at 10⁸ spores/mL, while Beauveria bassiana and Metarhizium anisopliae showed comparatively lower mortality rates. Smitha (2007) observed that as the fungal solution was diluted, mortality rates decreased. Additionally, the seventh-day infection of Anagasta craccivora resulted in 91% and 93% mortality rates, a trend also observed in this study. Beauveria bassiana spore concentration at 1.2 × 10⁴ spores/mL and Metarhizium anisopliae at 2.45 × 10⁶ spores/mL were examined by Chandler et al. (2005). Dogan et al. (2017) reported that Metarhizium brunneum (strains ARSEF 4556 and V275), Lecanicillium lecanii (strain UPH-0241), and Beauveria bassiana (strain UPH-1103) successfully reduced the population of two-spotted spider mites by 57.3% to 90.3% when exposed to a concentration of 1 × 10⁷ conidia/mL under controlled conditions in Petri dishes. AgBioForum, 26(3), 2024 | 153 Xuan et al — Biocontrol Potential of Different Entomopathogenic Fungi and Neem Oil for Control of Sucking Insects and Two-Spotted... The results of this study confirmed the significant pathogenic effects of the entomopathogenic fungi, as evidenced by the low LC₅₀ values observed. However, a decrease in fungal spore concentration resulted in lower mortality rates. Previous studies have also reported low LC₅₀ values for entomopathogenic fungi, such as 1.2 × 10⁴ spores/mL for Brevicoryne brassicae and 2.7 × 10⁴ spores/mL for Aphis gossypii. Among the tested fungal isolates, Verticillium lecanii and Hirsutella thompsonii exhibited higher virulence, achieving an LC₅₀ of 2.5 × 10⁴ spores. Similar findings were reported by researchers who recorded an LT₅₀ of 3.17 days for Beauveria bassiana. Additionally, Saranya et al. (2010) found that V. lecanii induced mortality in Aphis fabae populations on bean crops within approximately 3.31 days. Combining three types of entomopathogenic fungi with neem oil resulted in the highest mortality rates against T. urticae, A. gossypii, and B. tabaci. Azadirachtin in neem oil inhibits molting and reduces feeding, facilitating fungal mycelium penetration into the insect’s chitin layer. The effectiveness of this combination is influenced by the chemical composition of neem oil, including phytoalexins, terpenoids, and triterpenoids, which affect the compatibility of EPFs. Ribeiro et al. (2012) noted that these changes can impact insect- pathogenic fungi, while Mohan et al. (2007) highlighted the role of genetic variability in fungal isolates' compatibility with plant protection products. A study demonstrated that a combination of Verticillium lecanii, Beauveria bassiana, and Azadirachtin reduced A. gossypii populations by 30% to 70%. Oil-based formulations of B. bassiana and Metarhizium anisopliae achieved a 96.67% mortality rate in leafhoppers, and V. lecanii caused mortality rates of 96.77% in aphids and 97% in thrips by day 10. Additionally, experiments showed no significant inhibition of B. bassiana growth when cultured with 5% neem seed extract. Farmers increasingly use EPF as a sustainable pest management approach in agriculture, providing an effective alternative to chemical pesticides. As natural organisms, EPF offer excellent insect pest control, contributing to food security and supporting sustainable agricultural practices. IPM benefits greatly from EPF, offering a natural solution to concerns about the adverse effects of synthetic pesticides on human health, biodiversity, and ecosystem balance. EPF are effective biological control agents as they naturally infect and eliminate pests. Liu, Smagghe, & Liu (2023) conducted a study that highlighted the importance of EPF- host molecular interactions, focusing on glycans as crucial components for fungal adhesion and infection. Understanding the biochemical processes involved is essential for developing targeted biocontrol methods that yield effective results. By studying the biological complexity of these interactions, researchers can create more efficient EPF-based biopesticides. The knowledge gained from this study supports the development of precision agriculture and enhances the understanding of pathogen-host dynamics in pest management. Trinh, Ha, & Qiu (2020) evaluated various EPF strains in laboratory conditions to assess their potential for controlling Megoura japonica bean aphids. They found fungal isolates that effectively eliminated aphid populations, highlighting the importance of native fungal strains for pest control. This research demonstrated the efficiency of EPF in developing pest management systems for aphid outbreaks in legume plants and other crops. Sani et al. (2020) investigated biological control for Bemisia tabaci, aligning with Trinh et al.'s findings. Their study showed that EPF is a valuable pest management tool, working well in various agricultural systems and in combination with other biocontrol agents like parasites and predators. They also explored different EPF formulation strategies and application methods, highlighting their role in eco-friendly pest management. Aravinthraju et al. (2024) built on previous studies by examining how endophytic EPF enhances plant resistance to pests. The research revealed that EPF colonises plant tissues, triggering defensive mechanisms against stress. This approach can be used in pest and disease management, combining endophytic plants with fast- fluorescing bacteria. Mantzoukas et al. (2022) investigated EPF's pest control abilities across agricultural systems. Their research confirmed that EPF functions through two mechanisms: pathogen infection and ecological changes, affecting soil microbial communities, plant growth, and pest behaviour. Further interdisciplinary research and funding are needed to fully integrate EPF as a sustainable pest management solution. Halder et al. (2021) studied the use of EPF combined with botanical extracts for managing sucking pests on okra in outdoor conditions. They found that organic farming and integrated pest management are most effective when their components are well-matched, prioritising ecosystem health. Their data supports the development of integrated biological and botanical pest control products for sustainable crop protection. Aquino-Bolaños et al. (2023) demonstrated that EPF in oil- based suspensions could effectively control the agave weevil, Scyphophorus acupunctatus. Laboratory tests showed that oil preparations helped fungi survive and attach to insect exoskeletons, increasing the likelihood of fungal infection. These findings are significant for agave- dependent regions, highlighting the potential of EPF as a sustainable, environmentally friendly pest control method. Du, Xia, & Jin (2022) used biotechnological methods to enhance EPF strains by modifying key transcription factor genes, including MaSom1. This led to increased virulence, conidial production, and environmental tolerance. Their research shows how genetic engineering can improve EPF as an effective biopesticide, while emphasising the need for proper regulation and risk assessment for genetically modified organisms in agriculture. Sharma, Sharma, & Yadav (2023) reviewed the role of EPF in sustainable agriculture, highlighting their benefits in pest management, pesticide reduction, soil quality improvement, and biodiversity protection. They suggest that EPF fungi should be incorporated into national and international agricultural strategies to address current ecological challenges. Their findings provide valuable resources for policymakers, researchers, and practitioners aiming to promote environmentally friendly agricultural systems. Ebani & Mancianti (2021) expanded their investigations into the veterinary domain, demonstrating the effectiveness AgBioForum, 26(3), 2024 | 154 Xuan et al — Biocontrol Potential of Different Entomopathogenic Fungi and Neem Oil for Control of Sucking Insects and Two-Spotted... of EPF in managing livestock parasites, such as flies and mites, when combined with beneficial bacteria. This combined approach underscores the versatility of EPF, positioning them as key elements in integrated health systems for both agricultural and animal husbandry practices, thereby providing novel opportunities for the development of sustainable biocontrol methods. In a separate study, Kutalmış et al. (2023) examined the phylogenetic relationships among various EPF species to evaluate their effectiveness in controlling the pest Tropinota hirta (syn. Epicometis hirta) and the fungal pathogen Venturia inaequalis, which causes apple scab. Their findings highlighted the importance of understanding evolutionary connections when selecting the most suitable biocontrol agents. This research illustrates how phylogenetic studies can aid in making informed decisions about pest management, leading to the creation of more efficient and ecologically responsible biocontrol strategies. The body of scientific literature surrounding EPF continues to expand, reinforcing their potential as eco-friendly alternatives to chemical pesticides. The critical role of EPF in shaping the future of global agriculture is becoming increasingly apparent, offering targeted solutions to pest management issues. Ongoing advancements in research on EPF’s biology, ecological interactions, and application techniques will be instrumental in realising their full potential within sustainable agricultural frameworks worldwide. Conclusion In conclusion, this study highlighted the effectiveness of neem oil and EPF in controlling TSSM, aphids, and whiteflies, particularly when applied at high concentrations. When used in combination at lower concentrations, these agents demonstrated notable potential in managing the populations of these pests. However, further investigation into optimal application rates and field spraying timings is essential to maximise the synergistic effects of these treatments for future practical applications. Acknowledgment We would like to acknowledge the support of the Technical Cooperation project "Building Capacity for Can Tho University to be an Excellent Institution of Education, Scientific Research and Technology Transfer" of JICA, which partly funded this study. Conflicts of Interest The authors declare that there are no conflicts of interest. References Abdel-Raheem, M., Saad, A., & Abdel-Rahman, I. (2021). Entomopathogenic Fungi on Fabae Bean Aphid, Aphis Craccivora (Koch)(Hemiptera: Aphididae). Rom Biotechnol Lett, 26(4), 2862-2868. doi: https://doi.org/10.25083/rbl/26.4/2862-2868 Ambethgar, V. (2009). Potential of Entomopathogenic Fungi in Insecticide Resistance Management (Irm): A Review. Journal of Biopesticides, 2(2), 177-193. doi: http://doi.org/10.57182/jbiopestic .2.2.177-193 Ansari, M., & Butt, T. (2011). Effects of successive subculturing on stability, virulence, conidial yield, germination and shelf‐life of entomopathogenic fungi. Journal of Applied Microbiology, 110(6), 1460-1469. doi: https://doi.org/10.1111/j.1365- 2672.2011.04994.x Aquino-Bolaños, T., Ortiz-Hernández, Y. D., Bautista- Cruz, A., & Acevedo-Ortiz, M. A. (2023). Viability of Entomopathogenic Fungi in Oil Suspensions and Their Effectiveness against the Agave Pest Scyphophorus acupunctatus under Laboratory Conditions. Agronomy, 13(6), 1468. doi: https://doi.org/10.3390/agronomy13061468 Aravinthraju, K., Shanthi, M., Murugan, M., Srinivasan, R., Maxwell, L. A., Manikanda Boopathi, N., & Anandham, R. (2024). Endophytic Entomopathogenic Fungi: Their Role in Enhancing Plant Resistance, Managing Insect Pests, and Synergy with Management Routines. Journal of Fungi, 10(12), 865. doi: https://doi.org /10.3390/jof10120865 Bitsadze, N., Jaronski, S., Khasdan, V., Abashidze, E., Abashidze, M., Latchininsky, A., et al. (2013). Joint action of Beauveria bassiana and the insect growth regulators diflubenzuron and novaluron, on the migratory locust, Locusta migratoria. Journal of Pest Science, 86, 293-300. doi: https://doi.org/10.1007/s10340-012-0476-4 Bugeme, D. M., Knapp, M., Boga, H. I., Ekesi, S., & Maniania, N. K. (2014). Susceptibility of developmental stages of Tetranychus urticae (Acari: Tetranychidae) to infection by Beauveria bassiana and Metarhizium anisopliae (Hypocreales: Clavicipitaceae). International Journal of Tropical Insect Science, 34(3), 190-196. doi: https://doi.org/10.1017/S1742758414000381 Cakmak, I., Janssen, A., Sabelis, M. W., & Baspinar, H. (2009). Biological control of an acarine pest by single and multiple natural enemies. Biological Control, 50(1), 60-65. doi: https://doi.org/10 .1016/j.biocontrol.2009.02.006 Chandler, D., Davidson, G., & Jacobson, R. (2005). Laboratory and glasshouse evaluation of entomopathogenic fungi against the two-spotted spider mite, Tetranychus urticae (Acari: Tetranychidae), on tomato, Lycopersicon esculentum. Biocontrol Science and Technology, 15(1), 37-54. doi: https://doi.org/10.1080/09583150410001720617 Deguine, J.-P., Aubertot, J.-N., Bellon, S., Côte, F., Lauri, P.-E., Lescourret, F., et al. (2023). Agroecological crop protection for sustainable agriculture. Advances in Agronomy, 178, 1-59. doi: https://doi.org/10.1016/bs.agron.2022.11.002 Dogan, Y. O., Hazir, S., Yildiz, A., Butt, T. M., & Cakmak, I. (2017). Evaluation of entomopathogenic fungi for the control of Tetranychus urticae (Acari: Tetranychidae) and the effect of Metarhizium brunneum on the predatory mites (Acari: https://doi.org/10.25083/rbl/26.4/2862-2868 http://doi.org/10.57182/jbiopestic.2.2.177-193 http://doi.org/10.57182/jbiopestic.2.2.177-193 https://doi.org/10.1111/j.1365-2672.2011.04994.x https://doi.org/10.1111/j.1365-2672.2011.04994.x https://doi.org/10.3390/agronomy13061468 https://doi.org/10.3390/jof10120865 https://doi.org/10.3390/jof10120865 https://doi.org/10.1007/s10340-012-0476-4 https://doi.org/10.1017/S1742758414000381 https://doi.org/10.1016/j.biocontrol.2009.02.006 https://doi.org/10.1016/j.biocontrol.2009.02.006 https://doi.org/10.1080/09583150410001720617 https://doi.org/10.1016/bs.agron.2022.11.002 AgBioForum, 26(3), 2024 | 155 Xuan et al — Biocontrol Potential of Different Entomopathogenic Fungi and Neem Oil for Control of Sucking Insects and Two-Spotted... Phytoseiidae). Biological Control, 111, 6-12. doi: https://doi.org/10.1016/j.biocontrol.2017.05.001 Du, Y., Xia, Y., & Jin, K. (2022). Enhancing the Biocontrol Potential of the Entomopathogenic Fungus in Multiple Respects via the Overexpression of a Transcription Factor Gene MaSom1. Journal of Fungi, 8(2), 105. doi: https://doi.org/10.3390/jof8020105 Ebani, V. V., & Mancianti, F. (2021). Entomopathogenic Fungi and Bacteria in a Veterinary Perspective. Biology, 10(6), 479. doi: https://doi.org/10.3390 /biology10060479 Gatarayiha, M. C., Laing, M. D., & Miller, R. M. (2010). Effects of adjuvant and conidial concentration on the efficacy of Beauveria bassiana for the control of the two spotted spider mite, Tetranychus urticae. Experimental and Applied Acarology, 50, 217-229. doi: https://doi.org/10.1007/s10493-009-9307-6 González-Mas, N., Sánchez-Ortiz, A., Valverde-García, P., & Quesada-Moraga, E. (2019). Effects of Endophytic Entomopathogenic Ascomycetes on the Life- History Traits of Aphis gossypii Glover and Its Interactions with Melon Plants. Insects, 10(6), 165. doi: https://doi.org/10.3390/insects10060165 Halder, J., Divekar, P. A., & Rani, A. (2021). Compatibility of entomopathogenic fungi and botanicals against sucking pests of okra: an ecofriendly approach. Egyptian Journal of Biological Pest Control, 31, 1-7. doi: https://doi.org/10.1186/s41938-021-00378-6 Kutalmış, A., Terzioğlu, Z., Şen, R. H., & Sevim, A. (2023). Phylogenetic analysis and biocontrol potential of entomopathogenic fungi against Tropinota (= Epicometis) hirta (Poda)(Coleoptera: Cetoniidae) and the apple scab disease Ventura inaequalis. Egyptian Journal of Biological Pest Control, 33(1), 5. doi: https://doi.org/10.1186/s41938-023-00652-9 Liu, D., Smagghe, G., & Liu, T.-X. (2023). Interactions between Entomopathogenic Fungi and Insects and Prospects with Glycans. Journal of Fungi, 9(5), 575. doi: https://doi.org/10.3390/jof9050575 Mantzoukas, S., Kitsiou, F., Natsiopoulos, D., & Eliopoulos, P. A. (2022). Entomopathogenic Fungi: Interactions and Applications. Encyclopedia, 2(2), 646-656. doi: https://doi.org/10.3390/encyclopedia2020044 Mohan, M. C., Reddy, N. P., Devi, U. K., Kongara, R., & Sharma, H. C. (2007). Growth and insect assays of Beauveria bassiana with neem to test their compatibility and synergism. Biocontrol Science and Technology, 17(10), 1059-1069. doi: https://doi.org/10.1080/09583150701714551 Ribeiro, L., Blume, E., Bogorni, P., Dequech, S., Brand, S., & Junges, E. (2012). Compatibility of Beauveria bassiana commercial isolate with botanical insecticides utilized in organic crops in southern Brazil. Biological Agriculture & Horticulture, 28(4), 223-240. doi: https://doi.org/10.1080/01448765.2012.7350 88 Sani, I., Ismail, S. I., Abdullah, S., Jalinas, J., Jamian, S., & Saad, N. (2020). A Review of the Biology and Control of Whitefly, Bemisia tabaci (Hemiptera: Aleyrodidae), with Special Reference to Biological Control Using Entomopathogenic Fungi. Insects, 11(9), 619. doi: https://doi.org/10.3390/insects11090619 Saranya, S., Ushakumari, R., Jacob, S., & Philip, B. M. (2010). Efficacy of different ntomopathogenic fungi against cowpea aphid, Aphis craccivora (Koch). Journal of Biopesticides, 3, 138-142. doi: https://doi.org/10.57182/jbiopestic.3.1.138-142 Sharma, A., Sharma, S., & Yadav, P. K. (2023). Entomopathogenic Fungi and Their Relevance in Sustainable Agriculture: A Review. Cogent Food & Agriculture, 9(1), 2180857. doi: https://doi.org /10.1080/23311932.2023.2180857 Smitha, M. S. (2007). Biology and management of root mealy bug on banana cultivars (Doctoral dissertation, Kerala Agricultural University, Vellanikkara). Retrieved from https://krishikosh. egranth.ac.in/handle/1/5810041463 Trinh, D. N., Ha, T. K. L., & Qiu, D. (2020). Biocontrol potential of some entomopathogenic fungal strains against bean aphid Megoura japonica (Matsumura). Agriculture, 10(4), 114. doi: https://doi.org/10.3390/agriculture10040114 Ullah, M. S., & Lim, U. T. (2015). Laboratory bioassay of Beauveria bassiana against Tetranychus urticae (Acari: Tetranychidae) on leaf discs and potted bean plants. Experimental and Applied Acarology, 65, 307-318. doi: https://doi.org/10.1007/s10493-014- 9871-2 Van der Sluijs, J. P., Simon-Delso, N., Goulson, D., Maxim, L., Bonmatin, J.-M., & Belzunces, L. P. (2013). Neonicotinoids, bee disorders and the sustainability of pollinator services. Current Opinion In Environmental Sustainability, 5(3-4), 293-305. doi: https://doi.org/10.1016/j.cosust.2013.05.007 Van Mele, P., Cuc, N. T. T., & Van Huis, A. (2010). Farmers' knowledge, perceptions and practices in mango pest management in the Mekong Delta, Vietnam. International Journal of Pest Management, 47(1), 7- 16. doi: https://doi.org/10.1080/09670870150215559 Van Mele, P., Hai, T., Thas, O., & Van Huis, A. (20010). Influence of pesticide information sources on citrus farmers' knowledge, perception and practices in pest management, Mekong Delta, Vietnam. International Journal of Pest Management, 48(2), 169-177. doi: https://doi.org /10.1080/09670870210139304 Żyła, D., Homan, A., & Wegierek, P. (2017). Polyphyly of the extinct family Oviparosiphidae and its implications for inferring aphid evolution (Hemiptera, Sternorrhyncha). PLoS One, 12(4), e0174791. doi: https://doi.org/10.1371/journal.pone.0174791 https://doi.org/10.1016/j.biocontrol.2017.05.001 https://doi.org/10.3390/jof8020105 https://doi.org/10.3390/biology10060479 https://doi.org/10.3390/biology10060479 https://doi.org/10.1007/s10493-009-9307-6 https://doi.org/10.3390/insects10060165 https://doi.org/10.1186/s41938-021-00378-6 https://doi.org/10.1186/s41938-023-00652-9 https://doi.org/10.3390/jof9050575 https://doi.org/10.3390/encyclopedia2020044 https://doi.org/10.1080/09583150701714551 https://doi.org/10.1080/01448765.2012.735088 https://doi.org/10.1080/01448765.2012.735088 https://doi.org/10.3390/insects11090619 https://doi.org/10.57182/jbiopestic.3.1.138-142 https://doi.org/10.1080/23311932.2023.2180857 https://doi.org/10.1080/23311932.2023.2180857 https://krishikosh.egranth.ac.in/handle/1/5810041463 https://krishikosh.egranth.ac.in/handle/1/5810041463 https://doi.org/10.3390/agriculture10040114 https://doi.org/10.1007/s10493-014-9871-2 https://doi.org/10.1007/s10493-014-9871-2 https://doi.org/10.1016/j.cosust.2013.05.007 https://doi.org/10.1080/09670870150215559 https://doi.org/10.1080/09670870210139304 https://doi.org/10.1080/09670870210139304 https://doi.org/10.1371/journal.pone.0174791