A mycoinsecticide from Metarhizium anisopliae (Ascomycota: Hypocreales) based on a solid-state fermentation method against some aphid species in Türkiye

A mycoinsecticide from Metarhizium anisopliae (Ascomycota: Hypocreales) based on a solid-state fermentation method against some aphid species in Türkiye | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article A mycoinsecticide from Metarhizium anisopliae (Ascomycota: Hypocreales) based on a solid-state fermentation method against some aphid species in Türkiye Seda Biryol, İsmail Demir This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3314382/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 08 Apr, 2026 Read the published version in International Journal of Tropical Insect Science → Version 1 posted 5 You are reading this latest preprint version Abstract The research aims to propose an appropriate and efficient mycoinsecticide from a local Metarhizium anisopliae (Ascomycota: Hypocreales) strain (KTU-51) against several aphid species (Hemiptera: Aphididae; Myzus persicae Sulz., Aphis fabae Scop., Brevicoryne brassicae L. and Macrosiphum rosae L.), which are the most important agricultural pests. M. anisopliae produced spores in large quantities utilizing solid-state fermentation (SSF) with rice as a substrate. An oil-based mycoinsecticide called AFIDISIDAL-OD Met-TR61 was improved by incorporating spores harvested from the sporulated biomass. The product provided a more deathful effect than commercial products against all the aphis species under laboratory conditions. Met -TR61 with 10 8 spores/ml concentration yielded 78.3% mortality in the leaf disc experiment and 79% in the pot experiment on Myzus persicae . Other aphids were also found to be extremely sensitive to the product. In this particular study, an oil-based mycoinsecticide was developed and its efficacy for biological control of aphid species was assessed. The study’s findings indicate that the mycoinsecticide has the potential to be a workable and effective alternative to conventional chemical insecticides for controlling aphid populations. Aphid biocontrol mycoinsecticide oil-based formulation solid-state fermentation Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Aphids (Hemiptera: Aphididae) are among the most important pest groups in agriculture and horticulture all over the world. They both cause direct damage by absorbing plant sap and are effective in spreading plant pathogens from one plant to another. By causing the formation of honeydew deposits on the surface of the plant leaves, they also cause the photosynthetic surface of the leaves to decrease (Blackman and Eastop 2007; Hogenhout et al. 2008 ). It is known that 15 aphid species are very important pests for agricultural areas worldwide (Van Emden and Harrington 2017). Among these species, is Myzus persicae Sulz. and Aphis fabae Scop. are emphasized as essential pests. Brevicoryne brassicae L., which primarily feeds on the above-ground parts of its host plants, has been known to cause significant direct and indirect damage, ultimately resulting in production losses ranging from 35–80%, according to Ramanujam et al. ( 2017 ). The rose aphid, Macrosiphum rosae L., is an important aphid species that infests and damages roses (Karlik and Tjosvold 2003 ). According to Barbosa ( 1998 ), a variety of pest management strategies have been used including agricultural practices, mechanical, physical, chemical and biological control. Aphids are primarily managed by insecticides at the moment (Foster et al. 2007 ). The uncontrolled use of insecticides has resulted in issues such as pesticide resistance, pest comeback, negative impacts on biocontrol agents, environmental contamination, and hazardous residue buildup in the natural ecosystem (Silva et al. 2012 ). Biological control is an alternative method of pest control. Aphids have many natural enemies that can be used for biological control, such as predators, parasites, and pathogens (Bloemhard and Ramakers 2008). In recent years, a large number of studies have been conducted on the effectiveness of microbial pathogens including viruses, bacteria, and fungi an important agricultural pests (Ince et al. 2007 ; Secil et al. 2012 ; Sevim et al. 2012 ; Kocacevik et al. 2015 ; Eski et al. 2018 ; Gencer et al. 2020 , Usta 2022 ). Many biological control products developed from these microorganisms are used as pesticides in integrated pest management (IPM) programs against various pests, including aphids (Vega et al. 2008). Nearly 1000 entomopathogenic fungi, known to be effective in maintaining the natural balance of insect populations, have been reported (Sandhu et al. 2012 ). A significant number of these species appear to have a high potential to be developed as mycoinsecticide and mycoacaricide and used in the biological control of pests (de Faria and Wraight 2007 ; Khan et al. 2012 ). In recent studies, entomopathogenic fungi (EPF) have been shown to be among the best biocontrol agents for aphids. Especially entomopathogenic fungal pesticides have critical importance in IPM applications because these organisms cause diseases in insects and suppress their growth and rate of multiplication (Thomas and Read 2007 ). They have considerable advantages compared to conventional pesticides, notably cost-effectiveness, high yield, no negative impacts on beneficial organisms, no chemical residues in the environment, and increasing biodiversity in ecosystems (Vega et al. 2009 ; Lacey 2016 ). The most common species used against aphids are Lecanicillium spp., Beauveria spp., Metarhizium spp., and Isaria fumosorosea (formerly Paecylomyces fumosoroseus ), which belong to the order Hypocreales of the division Ascomycota (de Faria and Wraight 2007 ; Khan et al. 2012 ). Various entomopathogenic fungi, such as, Metarhizium anisopliae , Beauveria bassiana and others, have been reported as substantially control agents of several aphid species such as Myzus persicae (Sulzer), Aphis gossypii (Glover), Melanocallis caryaefoliae (Davis), Aulacorthum solani (Kaltenbach) (Kim et al. 2007; Shapiro-Ilan et al. 2008 ; Mohammed et al. 2018 ). Under unfavorable climatic circumstances, formulations can greatly enhance the field efficacy of entomopathogenic fungal products while also protecting their activity and persistence (Jackson et al. 2010 ). The approach recognized for avoiding abiotic factors is an oil-based formulation so that they can prevent conidia from imbibitional damage, the harmful impact of Uv irradiation, or chemical pesticides (Alves et al. 1998 ; Lopes et al. 2011 ). As a result, using oil formulations containing EPFs may enhance conidial stability and prolong their durability in the field, protecting fungi against heat stress, water loss, and, in importantly, UV radiation (Barreto et al. 2016 ). EPFs differ from other insect pathogens since they can infect through the hosts' integument. EPF infections are not limited to chewing insects, as they are not required to be ingested or eaten. They are unique in controlling insect pests that feed by sucking plant or animal juices (Lacey and Goettel 1995 ; Biryol et al. 2021 ; Eski et al. 2022 ). Despite all of the control methods, strategies and agents, the aphid species continue to be a major agricultural and horticultural pest, and their harm is gradually increasing. Thus, the objective of this study is to develop a local mycoinsecticide from Metarhizum anisopliae (KTU-51) that originated from soil samples against the different aphid species in Türkiye. Materials and Methods Providing and rearing the aphids Myzus persicae colonies were obtained from the Central Plant Protection Research Institute (Ankara, Türkiye) and were maintained on pepper seedlings ( Capsicum annuum ). Pepper seedlings were grown in pots. Growing conditions and temperatures of 25 ± 2°C (L) and 18 ± 2°C (D) were maintained in a 65% RH plant growth chamber (Biryol et al. 2022 ). Aphis fabae , Brevicoryne brassicae and Macrosiphum rosae were collected from infested areas in Trabzon, Türkiye and cultured on bean ( Phaseolus vulgaris L.), cabbage ( Brassica oleracea capitata ), and rose ( Rosa sp.) plant under laboratory conditions, respectively. These aphids were maintained on bean leaves, cabbage leaves, and fresh rose shoots, respectively. Experiments were performed on the same materials. The aphid culture was maintained until all experiments were completed. Fungal cultures Metarhizium anisopliae (KTU-51) was obtained from the entomopathogenic culture collection of Karadeniz Technical University, Faculty of Science, Department of Biology, Microbiology Laboratory. Previously, Biryol et al. ( 2022 ) revealed that this isolate had an effect of over 80% on M. persicae . Considering its effectiveness, KTU-51 has the potential to be developed as a mycoinsecticide and used in the biological control of aphids. Spore suspensions To maintain stock cultures, sterile cryovials containing 10% glycerol (Sigma-Aldrich) in sterile 0.01% Tween 80 solution were utilized and stored at -80°C until they were required for experiments. A 10-day incubation period on Sabouraud dextrose agar with yeast (SDAY) at 25 ± 2°C was used to cultivate fungal cultures and promote sporulation. Conidia were harvested by gently scraping and suspending them in a 0.01% Tween 80 solution. The spore concentration was established and adjusted for test concentrations by filtering the conidial suspension with sterile muslin and homogenizing it by vortexing for 2 min. The conidium viability test was performed according to the method of Hywell-Jones and Gillespie (1990). After 24 hours, the ratio of germinated spores on each plate was evaluated using a microscope, with positive germination defined as germ tube length that is at least half the spore length. All isolates exhibited a viability rate of more than 90%. Development of the SSF The mycoinsecticide was developed as an oil formulation using KTU-51 obtained from a solid substrate. The strain was first cultivated in a liquid medium and later transferred to a solid substrate. Afterward, spores were collected from the PDA medium as described above. The spore suspension was counted with a Neubauer hemocytometer, and the concentration was adjusted to 2.2 x 10 6 conidia/ml. To produce blastospores, a spore suspension of 1 ml was added to a liquid medium (containing 30 g glucose, 25 g casein hydrolysate, 20 g yeast extract, 4 g potassium hydrogen phosphate (K2HPO4) and 10 mg gentamicin per liter) in a ratio of 1:10. The resulting mixture was placed in a 500 ml flask, with 150 ml volume, and incubated at 28°C and 150 rpm for a period of 4 days, as reported by Seema et al. ( 2013 ). The pH of the medium was maintained at 5.6. Following inoculation, the liquid culture comprising blastopores was centrifuged for one minute at 10,000 rpm, and the spores were dissolved in sterile distilled water. The concentration was determined to be 1 × 10 7 − 5 × 10 8 conidia/ml The rice was used as a SS (solid substrate) to provide both a source of carbon and energy. Semi-cooked rice removed from its starch was distributed in growth bags had air holes of 150 gr per bag and autoclaved at 121°C at 1.1 atm pressure for 40 minutes (Seema et al. 2013 ). After the rice bags had cooled to room temperature, a 7.5 ml suspension of blastopores was inoculated into each bag, and fungal spores were brought into contact with rice grains by massaging. For growth, they were kept in an incubator at 25°C for 20–30 days. For regular and stable fungal growth during the incubation, the fungus growth bags were massaged every 5 days, and the rice-fungus mixtures became homogeneous. The rice was covered with fungal bodies placed in Kraft paper bags for 10 days to pre-dry. The fungus spores were then removed from the rice surface with a sieve (45 µm mesh − 1 ) and dried in a vacuum desiccator until the humidity level was less than 5%. A suspension of the sieved conidia was obtained by adding 1 g of conidia to 50 ml of sterile distilled water. A 100 µl of the decimal dilutions of the solution was inoculated into Sabouraud Dextrose Agar and spread with a glass spreader. The resulting CFUs were counted at 72 h post-inoculation at 28°C. The calculation was performed according to dilution with the appropriate number of colonies (30–300 colonies). Finally, spores were individually packaged in 100 ml glass bottles, and stored in a refrigerator with low humidity capacity at + 4°C until used in oil formulations (Fig. 1 ). To prolong the shelf life of the fungi before application, an oil-based formulation was developed according to Jackson et al. ( 1997 ). The oil-based formulation was prepared as described by Nian et al. ( 2015 ) and Biryol et al ( 2021 ) with minor modifications. The formulation was prepared by mixing naphthalene-2-sulfonic acid (3%), lecithin (12%), silvet L-77 (1%), ascorbic acid (0.1%), sodium alginate (1%), and vegetable oil (40%) with 10 gr powder spores (10 10 spore/g) to protect its physical and chemical properties, to extend its permanence and shelf life in nature. The mixture was then diluted to 100 ml using sterile distilled water and stirred gently (at 50 rpm) for 5 minutes at a temperature below 10°C until it became homogenous, which took approximately 30 minutes. A local new mycoinsecticide was named AFIDISIDAL-OD Met -TR61 (Biryol et al. 2021 ; 2022 ) (Fig. 1 ). Insecticidal effect of the mycoinsecticide on Myzus persicae An oil-based mycoinsecticide (AFISIDAL-OD Met -TR61), developed from Metarhizium anisopliae acting on Myzus persicae , was tested against the aphid using both leaf disc and pot assays. A concentration of 1 × 10 8 conidia/ml was applied by diluting the products at a 1:10 ratio. A commercial product (MET52) including M. anisopliae was used as a positive control. Untreated groups used as control were also created as the blank product, 0.01% Tween 80 without fungal spores. The leaf disc experiment was conducted on pepper leaves on water agar in Petri dishes. A paintbrush transferred fifty M. persicae nymphs with five repetitions to the discs. The mycoinsecticide was applied to Petri dishes with a hand sprayer. Petri dishes were placed in Plexiglas cages (35 × 40 × 35 cm) and maintained at 25 ± 1°C, ≥ 65% RH, and a photoperiod of 12:12 (L:D) h. Mortality rates were recorded after 1, 3, 5, and 7 days post-infection. After surface-sterilizing cadavers with 0.2% sodium hypochlorite solution and washing them three times in sterile distilled water, fungal emergence and sporulation on the cadavers were observed by placing them in a moisture chamber to encourage sporulation outside the cadavers. The pot experiment on M. persicae was carried out on healthy pepper seedlings according to Biryol et al. ( 2022 ). Selected seedlings were planted in pots with a 30 cm diameter and waited until the seedlings had at least seven leaves. Once the required growth was observed, the M. persicae nymphs were released into the pots, and populations were allowed to develop until there were about fifty per plant with five repetitions. The mycoinsecticide was sprayed on the seedlings as 1 ml with a concentration of 1 x 10 8 conidia/ ml. The pot experiment was conducted at 25 ± 1°C, 65 ± 5% relative humidity, and a light: dark cycle of 16:8 h in a climate room. For 14 days, the application was monitored. Dead insects were recorded and tested for mycosis using a moisture cycle. Host range of the mycoinsecticide The leaf disc assay was used to determine the host distribution of the mycoinsecticide on three other aphids ( Aphis fabae , Brevicoryne brassicae and Macrosiphum rosae ) that caused significant economic losses on crops or plants. In the host range experiment, tests were conducted on bean leaves for A. fabae , cabbage leaves for B. brassicae , and rose leaves for M. rosae on water agar in Petri dishes. Fifty nymphs with five repetitions were transferred to the discs using a paintbrush. The mycoinsecticide was applied to Petri dishes with a hand sprayer. Experiments were carried out using conditions in leaf disc application. The mortality values of the insecticide on aphids and the mycosis rates of the cadavers were calculated at the end of the application. Statistical analysis Abbott's (1925) method was used to correct the mortality statistics, and the % mycosis results for the fungal bioassays were determined using mycelia growth outside the cadaver. To compare test isolates to each other and the control group in terms of mortality and mycosis (for the fungal bioassays), the results were submitted to ANOVA, followed by LSD multiple comparison tests (p < 0.05). SPSS 28.0 was used to compute all experiments, and GraphPad Prism was used to construct the drowning graph (version 6.0, GraphPad Software, USA). Results Laboratory and pot experiment of the Met -TR61 on M. persicae The oil-based mycoinsecticide AFISIDAL-OD Met -TR61 was produced from powder spores containing 10 9 spores per ml as mycoinsecticides of M . anisopliae KTU-51 strain. The viability rate was determined to be 95%, fluid, and dark green in color (Fig. 1 ). Insecticidal efficacy experiments showed that Met -TR61 had a significant lethal effect on M. persicae in both leaf disc and pot experiments (p < 0.05) (Table 1 ). In the leaf disc, it was determined that the deaths that started on the third day after the infection increased with the prolongation of the period (Fig. 2 ). Met -TR61 with 10 8 spores/ml concentration yielded 44.3, 66.3 and 78,3% mortality on 3rd, 5th and 7th post-infection, respectively (F (6,24) ) = 22.60, df = 3, p < 0.05). As a result of the humidity circle application, 92.6% of cadavers had mycosis (p < 0.05). The commercial product (MET52) produced lower mortality than Met -TR61 on the 3rd, 5th, and 7th days after infection when death numbers were recorded (p < 0.05, Table 1 ). Only 43.6% of the cadavers were found to have mycosis for commercial products (p < 0.05). Under the same conditions and time, the blank product genetated 19.6% of M. persicae (p < 0.05). Mortality rates in the control group were 17.6% of M. persicae after the 7th-day post-treatment (p < 0.05) (Fig. 2 ). Table 1 Susceptibility of Myzus persicae nymphs exposed at 1 × 10 8 conidia/ml of Met -TR61 in leaf disc and pot experiment under laboratory conditions. Characters The leaf disc experiments The pot experiments Mortality% ±SE CI 95% Mycosis % CI 95% Mortality% ±SE CI 95% Mycosis % CI 95% Met -TR61 78.33 ± 1.15 a 73.36–83.29 92.66 ± 1.27 x 89.03-110.96 79.00 ± 1.52 A 72.42–85.57 81.33 ± 1.33 m 75.59–87.07 MET52 75.00 ± 2.88 b 62.57–87.42 43.66 ± 1.56 y 35.54–61.92 72.33 ± 1.52 B 65.75–78.90 76.33 ± 1.15 n 71.03–80.96 Blank product 19.69 ± 1.15 c 14.72–24.65 NG NS 16.33 ± 1.15 C 11.36–21.29 NG NS Control 18.32 ± 1.76 d 10.73–25.91 NG NS 10.66 ± 0.66 D 7.79–13.53 NG NS Note: Mortality% ± SE within a column followed by the upper case letter indicates the differences between mortality and lower case indicates the difference in the rate of mycosis after mortality (P < 0.05) Tukey’s LSD test, P ≤ 0.05). NS: Nonsignificant, NG: Non-growth fungus The pot experiment results revealed that plant vitality and flowering status were high and aphid populations were low in the seedlings where the Met -TR61 was applied (Fig. 3 ). On the contrary, plant viability and flowering status were incredibly low in the control group and the blank product, the seedlings died, and the number of aphids increased gradually in the first 3 days post-infection. Met -TR61 was determined as the most effective oil-based formulation in the pots experiment with 79% mortality (F (3,8) ) = 807.51, df = 3, p < 0.05) (Table 1 ). As a positive control, while MET52 caused 72,33% insecticidal effects on the pest, the blank product showed 16.3% mortality (p < 0.05). It was determined that commercial products on M. persicae nymphs were similar to each other in terms of mortality (p < 0.05). Mean aphid mortality in control was determined at 10.6% (p < 0.05). No fungal infection and mycosis were detected on aphids used in the blank product and control (p < 0,05) (Table 1 ). Effect of Met -TR on different aphid species Met -TR also produced remarkably high mortality values on other aphid species used in the study (F (3,8) ) = 1.28, df = 3, p < 0.05). Mortality rates were calculated as 79% on A. fabae , 84% on B. brassicae and 82% on M. rosae with 10 8 conidia/ml concentration on the 7th-day post-infection (F (3,11) = 4.003, p < 0.05), and mycosis rates of the cadavers were found as 84.0, 90.6 and 85.0%, respectively. The mortality values of the commercial product (MET52) on the same aphid species were determined to be 69% and 75% (p < 0.05). Also, the blank product was between 18% and 20% under the same conditions and time (p < 0.059 (Table 2 , Fig. 4 ). Table 2 Susceptibility of different aphid nymphs exposed at 1 × 10 8 conidia ml − 1 of Met -TR61 Characters Brevicoryne brassicae Aphis fabae Macrosiphum rosae Mortality% ±SE CI 95% Mortality% ±SE CI 95% Mortality% ±SE CI 95% Met -TR61 84.00 ± 1.15 A 79.03–88.96 79.00 ± 1.73 B 71.54–86.45 82.00 ± 1.15 A 77.03–86.96 MET52 73.33 ± 1.76 C 65.74–80.92 69.33 ± 2.60 D 58.13–80.53 71.00 ± 1.15 CD 66.03–75.96 Blank product 18.33 ± 2.02 E 9.60 ± 27.05 18.00 ± 1.73 E 10.54–25.45 18.33 ± 2.02 E 9.60 ± 27.05 Control 11.33 ± 1.76 F 3.74 ± 18.92 H 8.33 ± 1.45 G 2.08–14.58 9.33 ± 1.45 G 3.08 ± 15.58 Note: Mortality% ± SE within a column followed by the letter indicates the differences in mortality (P < 0.05) Tukey’s LSD test, P ≤ 0.05. In particular, the effect of the new product on B. brassicae was found to be higher and significantly different than A. fabae (p < 0.05) (Table 2 ). It was determined that the results on B. brassicae and M. rosea were close and there was no significant difference (p < 0.05). When we compared the effect of commercial and blank products on aphids, it was determined that there was a similar mortality rate in all insects and no significant difference between them, but a significant difference in the control group (p < 0.05). Moreover, the aphid populations increased rapidly in the control and the blank product applications. Discussion The main objective of biocontrol research is to develop novel, useful, and readily applicable mycoformulations for use against target pests. The main goal of this study is to create a new oil-based mycoinsecticide from a strain of Metarhizium anisopliae that is native to Türkiye to control the Myzus persicae aphid species as well as a few other aphid species. Fungi such as Lecanicillium spp., Beauveria bassiana , Metarhizium anisopliae , and Isaria spp. are entomopathogenic fungi that play significant roles in regulating insect populations. Many entomopathogenic fungi that were effective against various insects in our country and all over the world were isolated from insect and soil samples, characterized according to their molecular and morphological features and their potential for use in the biological control of agricultural pests was determined (Biryol et al. 2020 ; Eski et al. 2022 ; Kocacevik et al. 2015 ; 2016; Gurulingappa et al. 2011 ). M. anisopliae is the second most widely exploited entomopathogenic fungi in biocontrol studies next to B. bassiana , and it is known to attack more than 200 insect species belonging to different orders (Jitendra et al. 2012 ; Chen et al. 2014 ). Entomopathogenic hyphomycetes-based mycoinsecticides have been tested against sucking insects such as aphids in recent years, and their effectiveness against such pests has been demonstrated (Milner, 1997 ; Vandenberg et al. 2001 ). The global demand for biopesticides is also increasing day by day. Most of these mycoinsecticides are produced in America, Europe, and Asia (Faria and Wraight 2007 ). The mycoinsecticide AFIDISIDAL-OD Met -TR61 based on M. anisopliae developed in this study was produced using solid-substrate fermentation. Spore yield can also be increased with carbon and nitrogen sources such as rice, yeast extract, corn maceration liquid, or molasses, which are used as substrates or carriers (Talwar 2005 ). This method was preferred especially because of the high spore yield and some problems encountered in the production of strains of this species in a solid substrate. Spores obtained from production on a solid substrate can be used in different types of formulations, such as granular and spray (aqueous and oil-based) (Leland 2001 ). EPF-based biopesticides tend to be more effective when formulated in oil, as the conidia are evenly distributed on the insect cuticle and leaf surface. In contrast, aqueous spore formulations tend to remain as drops on the surface after application, which may reduce their efficacy (Inyang et al. 2000 ; Wraight et al. 2016 ). In bioassay studies, we determined that AFIDISIDAL-OD Met- TR61 remained active in host plants after application. The benefits of oil-based formulations for pest control, such as mortality and protection against adverse environmental effects, have been widely reported (Hedimbi et al. 2008 ; Lopes et al. 2011 ; Oliveira et al. 2018 ). It was determined that the aphids we used in the current study were sensitive to both the new product (AFIDISIDAL-OD Met -TR61) and the commercial product. Met -TR61 caused higher mortality than the commercial biopesticide products in our bioassay on the four aphid species. Oil-based carrier fungus conidia are more resilient to environmental stress factors, making them more resilient to environmental stress factors such as humidity, temperature, and UV radiation, according to research by Kaaya and Hassan ( 2000 ) and Batta ( 2003 ). This may extend the conidia's shelf life and vitality and raise the fungus's likelihood of spreading infections. In a study, Biryol et al. ( 2022 ) reported that the isolate used in the current study was resistant to environmental factors (UV-B and temperature) experiments and had high virulence. The results of the mortality experiments strongly support the development of biopesticides for the control of the four aphid species. In the study conducted by Mohammed and Hatcher ( 2016 ) on the 3rd stage M. persicae nymphs, it was determined that Mycotal caused 100% mortality with 1 × 10 10 conidia/ml concentration in laboratory conditions on the 9th day. In the current study, Met -TR61 yielded a mortality rate of 79% in 7th days in a pot experiment of 1 × 108 conidia /ml concentration. The study by Lopes and Faria ( 2019 ) assessed the effect of oil-based formulation on the coffee beetle species Hypothenemus hampei . According to the findings of the study, the oil formulation had a 20% more considerable mortality rate after six days than the pure conidia suspension at a concentration of 2 x 10 7 conidia/ml. According to the research findings, unformulated spore suspensions prepared in water, particularly oil-based formulations, are more effective when compared to the results of both laboratory and field applications of pests, and the reason for this effect is the spread and adhesion of the conidia on the hydrophobic cuticle of the host arthropod (David-Henriet et al. 1998 ). It has even been included in bioassays to enhance the mortality rate of Spodoptera litura and Helicoverpa armigera (Vimala Devi and Prasad 1996 ; Vimala Devi and Hari 2009 ). According to Albernaz et al. ( 2009 ), M. anisopliae formulations based on 10% sunflower oil showed increased ovicidal activity against Aedes aegypti . The oil has no impact on germination because of its low volatility and compatibility with environmental conditions (ULV). It has been demonstrated to improve M. anisopliae conidia efficacy (Albernaz et al. 2009 ; Bateman et al. 1998 ; Batta 2003 ). EPF-based formulations must be effective before the immature stages of these pests reach the reproductive age, which means that they are effective in a short time. The maturation period of M. persicae is 7.6 days, according to a study (Hong et al. 2019 ), and when we transplanted it into our study, it was determined that the new mycopesticide was effective on this pest from the second day, and the pest population decreased. Conclusion Previous research on Metarhizium anisopliae KTU-51, including isolation, identification, and insecticidal activity studies, has revealed that KTU-51 holds great promise for biological pest control. The results of this study indicate that the local mycoinsecticide developed from this isolate is also effective against other aphids, particularly M. persicae , and can be used successfully for the biological control of these important agricultural pests. Studies on the commercialization of an effective, environmentally friendly, and ecologically compatible AFIDISIDAL-OD Met- TR61 must be conducted as soon as possible. Declarations Acknowledgment The authors deeply appreciate the financial support of KTU-BAP (The Scientific Research Committee of Karadeniz Technical University) for the project FDK-2017-5896. Author contributions The study was designed by ID and SB, with SB carrying out the research. ID and SB contributed to the initial draft of the manuscript, with both authors involved in subsequent review and editing. All authors have read and approved the final version of the manuscript. Funding The Karadeniz Technical University KTU-BAP (The Scientific Research Committee) has funded this research for the project FDK-2017-5896. Availability of data and materials All data generated and analyzed during this study are indicated in the manuscript. Ethics approval and consent to participate Not applicable. 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Elsevier Science, Amsterdam Khan S et al (2012) Entomopathogenic fungi as microbial biocontrol agent. Mol Plant Breed 3(7). https://doi.org/10.5376/mpb.2012.03.0007 Kocacevik S et al (2015) Molecular characterization, virulence and horizontal transmission of Beauveria pseudobassiana from Dendroctonus micans (Kug.) (Coleoptera: Curculionidae). J Appl Entomol 139:381–389. https://doi.org/10.1111/jen.12181 Lacey LA, Goettel MS (1995) Current developments in microbial control of insect pests and prospects for the early 21st century. Entomophaga 40:3–27. https://doi.org/10.1007/BF02372677 Lacey LA (ed) (2016) Microbial Control of Insect and Mite Pests: From Theory to Practice, 1st edn. Academic Press, Cambridge, MA, USA Leland JE (2001) Environmental-stress tolerant formulations of Metarhizium anisopliae var. acridum for control of African desert locust ( Schistocerca gregaria ) (Doctoral dissertation, Virginia Polytechnic Institute and State University). Dissertation. Virginia: Faculty of Virginia Polytechnic. http://scholar.lib.vt.edu/theses/available/etd_12052001_115455/unrestricted/ JlelandDisertation. PDF [01 March 2023] Lopes RB, Faria M (2019) Influence of two formulation types and moisture levels on the storage stability and insecticidal activity of Beauveria bassiana . Biocontrol Sci Technol 29(5):437–450. https://doi.org/10.1080/09583157.2019.1566436 Lopes RB et al (2011) Protection of entomopathogenic conidia against chemical fungicides afforded by an oil-based formulation. Biocontrol Sci Technol 21(2):125–137. https://doi.org/10.1080/09583157.2010.534548 Milner RJ (1997) Prospects for biopesticides for aphid control. Biocontrol 42(1–2):227. https://doi.org/10.1007/BF02769900 Mohammed AA et al (2018) Selection of highly virulent entomopathogenic fungal isolates to control the greenhouse aphid species in Iraq. Egypt J Biol Pest Control 28:1–7. https://doi.org/10.1186/s41938-018-0079-3 Mohammed AA, Hatcher PE (2016) Effect of temperature, relative humidity and aphid developmental stage on the efficacy of the mycoinsecticide Mycotal® against Myzus persicae . Biocontrol Sci Technol 26(10):1379–1400. https://doi.org/10.1080/09583157.2016.1207219 Nian XG et al (2015) Evaluation of alternative Plutella xylostella control by two Isaria fumosorosea conidial formulations–oil-based formulation and wettable powder–combined with Bacillus thuringiensis . Pest Manag Sci 71(12):1675–1684. https://doi.org/10.1002/ps.3977 Oliveira DGP et al (2018) Increased tolerance of Beauveria bassiana and Metarhizium anisopliae conidia to high temperature provided by oil-based formulations. J Invertebr Pathol 151:151–157. https://doi.org/10.1016/j.jip.2017.11.012 Polar P et al (2005) Comparison of water, oils and emulsifiable adjuvant oils as formulating agents for Metarhizium anisopliae for use in control of Boophilus microplus . Mycopathol 160:151–157. https://doi.org/10.1007/s11046-005-0120-4 Prior C et al (1988) Infectivity of oil and water formulations of Beauveria bassiana (Deuteromycotina: Hyphomycetes) to the cocoa weevil pest Pantorhytes plutus (Coleoptera: Curculionidae). J Invertebr Pathol 52:66–72. https://doi.org/10.1016/0022-2011(88)90103-6 Ramanujam B et al (2017) Field evaluation of entomopathogenic fungi against cabbage aphid, Brevicoryne brassicae (L.) and their effect on coccinellid predator, Coccinella septempunctata (Linnaeus). J BiolControl 168–171. https://doi.org/10.18311/jbc/2017/16350 Sandhu SS et al (2012) Myco-Biocontrol of insect pests: Factors involved, mechanism, and regulation. J Pathog 2012:126819. https://doi.org/10.1155/2012/126819 Secil ES et al (2012) Isolation, characterization and virulence of bacteria from Ostrinia nubilali s (Lepidoptera: Pyralidae). Biologia 67(4):767–776. https://doi.org/10.2478/s11756-012-0070-5 Seema Y et al (2013) Mass production of entomopathogens Beauveria bassiana and Metarhizium anisopliae using rice as a substrate by diphasic liquid-solid fermentation technique. Int J Adv Biol Res 3:331–335 Sevim A et al (2012) A Novel cry 2Ab gene from the indigenous isolate Bacillus thuringiensis subsp. kurstaki . J Microbiol Biotechnol 22(1):137–144. https://doi.org/10.4014/jmb.1108.08061 Shapiro-Ilan DI et al (2008) Virulence of Hypocreales fungi to pecan aphids (Hemiptera: Aphididae) in the laboratory. J Invertebr Pathol 99:312–317. https://doi.org/10.1016/j.jip.2008.07.001 Silva AX et al (2012) Insecticide resistance mechanisms in the green peach aphid, Myzus persicae (Hemiptera: Aphididae) I: a transcriptomic survey. PLoS ONE 7(6):e36366. https://doi.org/10.1371/journal.pone.0036366 Talwar BH (2005) Isolation and characterization of entomopathogenic fungi and their effectiveness. Ph.D. Thesis, In Agricultural Entomology, Department of Agricultural Microbiology, Dharwad Thomas MB, Read AF (2007) Fungal bioinsecticide with a sting. Nat Biotechnol 25(12):1367–1368. https://doi.org/10.1038/nbt1207-1367 Usta M (2022) Local isolate of Bacillus thuringiensis (Berliner, 1915) (Bacteria: Bacillaceae) from Cydalima perspectalis (Walker, 1859) (Lepidoptera: Crambidae: Spilomelinae) includes cry1, cry3 and cry4 genes and their insecticidal activities. Turk J Entomol 46(2):227–237. https://doi.org/10.16970/entoted.1017243 Van Emden HF, Harrington R (2007) Aphids as crop pests, vol 717 ISBN–13. CABI, Cromwell Press, Trowbridge. 978 0 85199 819 0 Vandenberg JD et al (2001) Efficacy of fungi for control of Russian wheat aphid (Homoptera: Aphididae) in irrigated wheat. Southw Entomol 26:73–85 Vega FE et al (2009) Fungal entomopathogens: new insights on their ecology. Fungal Ecol 2(4):149–159. https://doi.org/10.1016/j.funeco.2009.05.001 Vimala Devi PS, Prasad YG (1996) Compatibility of oils and antifeedants of plant origin with the entomopathogenic fungus Nomuraea rileyi . J Invertebr Pathol 68:91–93. https://doi.org/10.1006/jipa.1996.0063 Vimala Devi PS, Hari PP (2009) Identification of a virulent isolate of the entomopathogenic fungus Beauveria bassiana (Balsamo) Vuillemin, its mass multiplication and formulation for development into a mycoinsecticide for management of Helicoverpa armigera (Hübner). J Biol Control 23:137–144 Wraight SP et al (2016) Efficacy of spray applications of entomopathogenic fungi against western flower thrips infesting greenhouse impatiens under variable moisture conditions. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3314382","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":278879858,"identity":"67ca013d-d970-437c-a222-9b1518e82dba","order_by":0,"name":"Seda Biryol","email":"","orcid":"","institution":"Trabzon University: Trabzon Universitesi","correspondingAuthor":false,"prefix":"","firstName":"Seda","middleName":"","lastName":"Biryol","suffix":""},{"id":278879859,"identity":"57a5af84-b53e-4cbe-8260-f24d8b608d92","order_by":1,"name":"İsmail Demir","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAvUlEQVRIiWNgGAWjYBACNoYEBiCyMQDzeEjQkmbAA9UiQYSmBBBxmAQtfOzJRzc83HPe2F4igfHB2zaGOvMGQg7jeZZ2I+HZbTMeiQRmw7ltDBIyBwhpkcgxu5Fw4LYNUAubNC9QC0GXsUnkfwNqOQfSwv6bSC05bEAtB0AOY2MmTgvPM5DDko15zjxslpxzTkJyBiEt8u3Jz27+OGBn2N6efPDDmzIbfmIiBgYYGxiIi8lRMApGwSgYBQQBAHH1N/zgQ0hVAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0001-6227-0039","institution":"Karadeniz Technical University: Karadeniz Teknik Universitesi","correspondingAuthor":true,"prefix":"","firstName":"İsmail","middleName":"","lastName":"Demir","suffix":""}],"badges":[],"createdAt":"2023-08-31 16:20:16","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3314382/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3314382/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s42690-026-01814-6","type":"published","date":"2026-04-08T15:58:24+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":52703495,"identity":"d079eeea-c6f9-48a0-a15c-7b5bfd212a22","added_by":"auto","created_at":"2024-03-14 18:10:28","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":516383,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"Fig.11.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3314382/v1/6ebf4812c4cd39920170f0e6.jpg"},{"id":52703496,"identity":"bc08c6d3-db23-457a-9319-39ca7ee3786f","added_by":"auto","created_at":"2024-03-14 18:10:28","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":325693,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"Fig.12.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3314382/v1/b8267ed6a1a6e13e423d2678.jpg"},{"id":52703498,"identity":"4db56469-a0e1-4f28-9c12-6346e7c6910b","added_by":"auto","created_at":"2024-03-14 18:10:28","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":415432,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"Fig.13.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3314382/v1/e0070ab65e42489a7f957420.jpg"},{"id":52703497,"identity":"a5403329-fa87-4b3e-a5d1-d262fc508d2d","added_by":"auto","created_at":"2024-03-14 18:10:28","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":284127,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"Fig.14.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3314382/v1/9c19d6e9fdd08df5e5b792d1.jpg"},{"id":106809632,"identity":"af59aa26-de67-4673-8985-ec6a5c08b800","added_by":"auto","created_at":"2026-04-13 16:11:56","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2403240,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3314382/v1/01cffef9-0e4d-46a1-b3f7-a6b07b91d3bc.pdf"}],"financialInterests":"","formattedTitle":"A mycoinsecticide from Metarhizium anisopliae (Ascomycota: Hypocreales) based on a solid-state fermentation method against some aphid species in Türkiye","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAphids (Hemiptera: Aphididae) are among the most important pest groups in agriculture and horticulture all over the world. They both cause direct damage by absorbing plant sap and are effective in spreading plant pathogens from one plant to another. By causing the formation of honeydew deposits on the surface of the plant leaves, they also cause the photosynthetic surface of the leaves to decrease (Blackman and Eastop 2007; Hogenhout et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). It is known that 15 aphid species are very important pests for agricultural areas worldwide (Van Emden and Harrington 2017). Among these species, is \u003cem\u003eMyzus persicae\u003c/em\u003e Sulz. and \u003cem\u003eAphis fabae\u003c/em\u003e Scop. are emphasized as essential pests. \u003cem\u003eBrevicoryne brassicae\u003c/em\u003e L., which primarily feeds on the above-ground parts of its host plants, has been known to cause significant direct and indirect damage, ultimately resulting in production losses ranging from 35\u0026ndash;80%, according to Ramanujam et al. (\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The rose aphid, \u003cem\u003eMacrosiphum rosae\u003c/em\u003e L., is an important aphid species that infests and damages roses (Karlik and Tjosvold \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2003\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAccording to Barbosa (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e1998\u003c/span\u003e), a variety of pest management strategies have been used including agricultural practices, mechanical, physical, chemical and biological control. Aphids are primarily managed by insecticides at the moment (Foster et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). The uncontrolled use of insecticides has resulted in issues such as pesticide resistance, pest comeback, negative impacts on biocontrol agents, environmental contamination, and hazardous residue buildup in the natural ecosystem (Silva et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Biological control is an alternative method of pest control. Aphids have many natural enemies that can be used for biological control, such as predators, parasites, and pathogens (Bloemhard and Ramakers 2008).\u003c/p\u003e \u003cp\u003eIn recent years, a large number of studies have been conducted on the effectiveness of microbial pathogens including viruses, bacteria, and fungi an important agricultural pests (Ince et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Secil et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Sevim et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Kocacevik et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Eski et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Gencer et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2020\u003c/span\u003e, Usta \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Many biological control products developed from these microorganisms are used as pesticides in integrated pest management (IPM) programs against various pests, including aphids (Vega et al. 2008). Nearly 1000 entomopathogenic fungi, known to be effective in maintaining the natural balance of insect populations, have been reported (Sandhu et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). A significant number of these species appear to have a high potential to be developed as mycoinsecticide and mycoacaricide and used in the biological control of pests (de Faria and Wraight \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Khan et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). In recent studies, entomopathogenic fungi (EPF) have been shown to be among the best biocontrol agents for aphids. Especially entomopathogenic fungal pesticides have critical importance in IPM applications because these organisms cause diseases in insects and suppress their growth and rate of multiplication (Thomas and Read \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). They have considerable advantages compared to conventional pesticides, notably cost-effectiveness, high yield, no negative impacts on beneficial organisms, no chemical residues in the environment, and increasing biodiversity in ecosystems (Vega et al. \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Lacey \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). The most common species used against aphids are \u003cem\u003eLecanicillium\u003c/em\u003e spp., \u003cem\u003eBeauveria\u003c/em\u003e spp., \u003cem\u003eMetarhizium\u003c/em\u003e spp., and \u003cem\u003eIsaria fumosorosea\u003c/em\u003e (formerly \u003cem\u003ePaecylomyces fumosoroseus\u003c/em\u003e), which belong to the order Hypocreales of the division Ascomycota (de Faria and Wraight \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Khan et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Various entomopathogenic fungi, such as, \u003cem\u003eMetarhizium anisopliae\u003c/em\u003e, \u003cem\u003eBeauveria bassiana\u003c/em\u003e and others, have been reported as substantially control agents of several aphid species such as \u003cem\u003eMyzus persicae\u003c/em\u003e (Sulzer), \u003cem\u003eAphis gossypii\u003c/em\u003e (Glover), \u003cem\u003eMelanocallis caryaefoliae\u003c/em\u003e (Davis), \u003cem\u003eAulacorthum solani\u003c/em\u003e (Kaltenbach) (Kim et al. 2007; Shapiro-Ilan et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Mohammed et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eUnder unfavorable climatic circumstances, formulations can greatly enhance the field efficacy of entomopathogenic fungal products while also protecting their activity and persistence (Jackson et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). The approach recognized for avoiding abiotic factors is an oil-based formulation so that they can prevent conidia from imbibitional damage, the harmful impact of Uv irradiation, or chemical pesticides (Alves et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Lopes et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). As a result, using oil formulations containing EPFs may enhance conidial stability and prolong their durability in the field, protecting fungi against heat stress, water loss, and, in importantly, UV radiation (Barreto et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). EPFs differ from other insect pathogens since they can infect through the hosts' integument. EPF infections are not limited to chewing insects, as they are not required to be ingested or eaten. They are unique in controlling insect pests that feed by sucking plant or animal juices (Lacey and Goettel \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e1995\u003c/span\u003e; Biryol et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Eski et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Despite all of the control methods, strategies and agents, the aphid species continue to be a major agricultural and horticultural pest, and their harm is gradually increasing. Thus, the objective of this study is to develop a local mycoinsecticide from \u003cem\u003eMetarhizum anisopliae\u003c/em\u003e (KTU-51) that originated from soil samples against the different aphid species in T\u0026uuml;rkiye.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eProviding and rearing the aphids\u003c/h2\u003e \u003cp\u003e \u003cem\u003eMyzus persicae\u003c/em\u003e colonies were obtained from the Central Plant Protection Research Institute (Ankara, T\u0026uuml;rkiye) and were maintained on pepper seedlings (\u003cem\u003eCapsicum annuum\u003c/em\u003e). Pepper seedlings were grown in pots. Growing conditions and temperatures of 25\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C (L) and 18\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C (D) were maintained in a 65% RH plant growth chamber (Biryol et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cem\u003eAphis fabae\u003c/em\u003e, \u003cem\u003eBrevicoryne brassicae\u003c/em\u003e and \u003cem\u003eMacrosiphum rosae\u003c/em\u003e were collected from infested areas in Trabzon, T\u0026uuml;rkiye and cultured on bean (\u003cem\u003ePhaseolus vulgaris\u003c/em\u003e L.), cabbage (\u003cem\u003eBrassica oleracea capitata\u003c/em\u003e), and rose (\u003cem\u003eRosa\u003c/em\u003e sp.) plant under laboratory conditions, respectively. These aphids were maintained on bean leaves, cabbage leaves, and fresh rose shoots, respectively. Experiments were performed on the same materials. The aphid culture was maintained until all experiments were completed.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eFungal cultures\u003c/h2\u003e \u003cp\u003e \u003cem\u003eMetarhizium anisopliae\u003c/em\u003e (KTU-51) was obtained from the entomopathogenic culture collection of Karadeniz Technical University, Faculty of Science, Department of Biology, Microbiology Laboratory. Previously, Biryol et al. (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) revealed that this isolate had an effect of over 80% on \u003cem\u003eM. persicae\u003c/em\u003e. Considering its effectiveness, KTU-51 has the potential to be developed as a mycoinsecticide and used in the biological control of aphids.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eSpore suspensions\u003c/h2\u003e \u003cp\u003eTo maintain stock cultures, sterile cryovials containing 10% glycerol (Sigma-Aldrich) in sterile 0.01% Tween 80 solution were utilized and stored at -80\u0026deg;C until they were required for experiments. A 10-day incubation period on Sabouraud dextrose agar with yeast (SDAY) at 25\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C was used to cultivate fungal cultures and promote sporulation. Conidia were harvested by gently scraping and suspending them in a 0.01% Tween 80 solution. The spore concentration was established and adjusted for test concentrations by filtering the conidial suspension with sterile muslin and homogenizing it by vortexing for 2 min. The conidium viability test was performed according to the method of Hywell-Jones and Gillespie (1990). After 24 hours, the ratio of germinated spores on each plate was evaluated using a microscope, with positive germination defined as germ tube length that is at least half the spore length. All isolates exhibited a viability rate of more than 90%.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eDevelopment of the SSF\u003c/h2\u003e \u003cp\u003eThe mycoinsecticide was developed as an oil formulation using KTU-51 obtained from a solid substrate. The strain was first cultivated in a liquid medium and later transferred to a solid substrate. Afterward, spores were collected from the PDA medium as described above. The spore suspension was counted with a Neubauer hemocytometer, and the concentration was adjusted to 2.2 x 10\u003csup\u003e6\u003c/sup\u003e conidia/ml. To produce blastospores, a spore suspension of 1 ml was added to a liquid medium (containing 30 g glucose, 25 g casein hydrolysate, 20 g yeast extract, 4 g potassium hydrogen phosphate (K2HPO4) and 10 mg gentamicin per liter) in a ratio of 1:10. The resulting mixture was placed in a 500 ml flask, with 150 ml volume, and incubated at 28\u0026deg;C and 150 rpm for a period of 4 days, as reported by Seema et al. (\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). The pH of the medium was maintained at 5.6. Following inoculation, the liquid culture comprising blastopores was centrifuged for one minute at 10,000 rpm, and the spores were dissolved in sterile distilled water. The concentration was determined to be 1 \u0026times; 10\u003csup\u003e7\u003c/sup\u003e \u0026minus;\u0026thinsp;5 \u0026times; 10\u003csup\u003e8\u003c/sup\u003e conidia/ml\u003c/p\u003e \u003cp\u003eThe rice was used as a SS (solid substrate) to provide both a source of carbon and energy. Semi-cooked rice removed from its starch was distributed in growth bags had air holes of 150 gr per bag and autoclaved at 121\u0026deg;C at 1.1 atm pressure for 40 minutes (Seema et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). After the rice bags had cooled to room temperature, a 7.5 ml suspension of blastopores was inoculated into each bag, and fungal spores were brought into contact with rice grains by massaging. For growth, they were kept in an incubator at 25\u0026deg;C for 20\u0026ndash;30 days. For regular and stable fungal growth during the incubation, the fungus growth bags were massaged every 5 days, and the rice-fungus mixtures became homogeneous.\u003c/p\u003e \u003cp\u003eThe rice was covered with fungal bodies placed in Kraft paper bags for 10 days to pre-dry. The fungus spores were then removed from the rice surface with a sieve (45 \u0026micro;m mesh\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and dried in a vacuum desiccator until the humidity level was less than 5%. A suspension of the sieved conidia was obtained by adding 1 g of conidia to 50 ml of sterile distilled water. A 100 \u0026micro;l of the decimal dilutions of the solution was inoculated into Sabouraud Dextrose Agar and spread with a glass spreader. The resulting CFUs were counted at 72 h post-inoculation at 28\u0026deg;C. The calculation was performed according to dilution with the appropriate number of colonies (30\u0026ndash;300 colonies). Finally, spores were individually packaged in 100 ml glass bottles, and stored in a refrigerator with low humidity capacity at +\u0026thinsp;4\u0026deg;C until used in oil formulations (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo prolong the shelf life of the fungi before application, an oil-based formulation was developed according to Jackson et al. (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e1997\u003c/span\u003e). The oil-based formulation was prepared as described by Nian et al. (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) and Biryol et al (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) with minor modifications. The formulation was prepared by mixing naphthalene-2-sulfonic acid (3%), lecithin (12%), silvet L-77 (1%), ascorbic acid (0.1%), sodium alginate (1%), and vegetable oil (40%) with 10 gr powder spores (10\u003csup\u003e10\u003c/sup\u003e spore/g) to protect its physical and chemical properties, to extend its permanence and shelf life in nature. The mixture was then diluted to 100 ml using sterile distilled water and stirred gently (at 50 rpm) for 5 minutes at a temperature below 10\u0026deg;C until it became homogenous, which took approximately 30 minutes. A local new mycoinsecticide was named AFIDISIDAL-OD \u003cem\u003eMet\u003c/em\u003e-TR61 (Biryol et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003eInsecticidal effect of the mycoinsecticide on\u003c/b\u003e \u003cb\u003eMyzus persicae\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAn oil-based mycoinsecticide (AFISIDAL-OD \u003cem\u003eMet\u003c/em\u003e-TR61), developed from \u003cem\u003eMetarhizium anisopliae\u003c/em\u003e acting on \u003cem\u003eMyzus persicae\u003c/em\u003e, was tested against the aphid using both leaf disc and pot assays. A concentration of 1 \u0026times; 10\u003csup\u003e8\u003c/sup\u003e conidia/ml was applied by diluting the products at a 1:10 ratio. A commercial product (MET52) including \u003cem\u003eM. anisopliae\u003c/em\u003e was used as a positive control. Untreated groups used as control were also created as the blank product, 0.01% Tween 80 without fungal spores.\u003c/p\u003e \u003cp\u003eThe leaf disc experiment was conducted on pepper leaves on water agar in Petri dishes. A paintbrush transferred fifty \u003cem\u003eM. persicae\u003c/em\u003e nymphs with five repetitions to the discs. The mycoinsecticide was applied to Petri dishes with a hand sprayer. Petri dishes were placed in Plexiglas cages (35 \u0026times; 40 \u0026times; 35 cm) and maintained at 25\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C, \u0026ge; 65% RH, and a photoperiod of 12:12 (L:D) h. Mortality rates were recorded after 1, 3, 5, and 7 days post-infection. After surface-sterilizing cadavers with 0.2% sodium hypochlorite solution and washing them three times in sterile distilled water, fungal emergence and sporulation on the cadavers were observed by placing them in a moisture chamber to encourage sporulation outside the cadavers.\u003c/p\u003e \u003cp\u003eThe pot experiment on \u003cem\u003eM. persicae\u003c/em\u003e was carried out on healthy pepper seedlings according to Biryol et al. (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Selected seedlings were planted in pots with a 30 cm diameter and waited until the seedlings had at least seven leaves. Once the required growth was observed, the \u003cem\u003eM. persicae\u003c/em\u003e nymphs were released into the pots, and populations were allowed to develop until there were about fifty per plant with five repetitions. The mycoinsecticide was sprayed on the seedlings as 1 ml with a concentration of 1 x 10\u003csup\u003e8\u003c/sup\u003e conidia/ ml. The pot experiment was conducted at 25\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C, 65\u0026thinsp;\u0026plusmn;\u0026thinsp;5% relative humidity, and a light: dark cycle of 16:8 h in a climate room. For 14 days, the application was monitored. Dead insects were recorded and tested for mycosis using a moisture cycle.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eHost range of the mycoinsecticide\u003c/h2\u003e \u003cp\u003eThe leaf disc assay was used to determine the host distribution of the mycoinsecticide on three other aphids (\u003cem\u003eAphis fabae\u003c/em\u003e, \u003cem\u003eBrevicoryne brassicae\u003c/em\u003e and \u003cem\u003eMacrosiphum rosae\u003c/em\u003e) that caused significant economic losses on crops or plants. In the host range experiment, tests were conducted on bean leaves for \u003cem\u003eA. fabae\u003c/em\u003e, cabbage leaves for \u003cem\u003eB. brassicae\u003c/em\u003e, and rose leaves for \u003cem\u003eM. rosae\u003c/em\u003e on water agar in Petri dishes. Fifty nymphs with five repetitions were transferred to the discs using a paintbrush. The mycoinsecticide was applied to Petri dishes with a hand sprayer. Experiments were carried out using conditions in leaf disc application. The mortality values of the insecticide on aphids and the mycosis rates of the cadavers were calculated at the end of the application.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAbbott's (1925) method was used to correct the mortality statistics, and the % mycosis results for the fungal bioassays were determined using mycelia growth outside the cadaver. To compare test isolates to each other and the control group in terms of mortality and mycosis (for the fungal bioassays), the results were submitted to ANOVA, followed by LSD multiple comparison tests (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). SPSS 28.0 was used to compute all experiments, and GraphPad Prism was used to construct the drowning graph (version 6.0, GraphPad Software, USA).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eLaboratory and pot experiment of the\u003c/b\u003e \u003cb\u003eMet\u003c/b\u003e\u003cb\u003e-TR61 on\u003c/b\u003e \u003cb\u003eM. persicae\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe oil-based mycoinsecticide AFISIDAL-OD \u003cem\u003eMet\u003c/em\u003e-TR61 was produced from powder spores containing 10\u003csup\u003e9\u003c/sup\u003e spores per ml as mycoinsecticides of \u003cem\u003eM\u003c/em\u003e. \u003cem\u003eanisopliae\u003c/em\u003e KTU-51 strain. The viability rate was determined to be 95%, fluid, and dark green in color (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eInsecticidal efficacy experiments showed that \u003cem\u003eMet\u003c/em\u003e-TR61 had a significant lethal effect on \u003cem\u003eM. persicae\u003c/em\u003e in both leaf disc and pot experiments (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). In the leaf disc, it was determined that the deaths that started on the third day after the infection increased with the prolongation of the period (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). \u003cem\u003eMet\u003c/em\u003e-TR61 with 10\u003csup\u003e8\u003c/sup\u003e spores/ml concentration yielded 44.3, 66.3 and 78,3% mortality on 3rd, 5th and 7th post-infection, respectively (F \u003csub\u003e(6,24)\u003c/sub\u003e)\u0026thinsp;=\u0026thinsp;22.60, df\u0026thinsp;=\u0026thinsp;3, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). As a result of the humidity circle application, 92.6% of cadavers had mycosis (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The commercial product (MET52) produced lower mortality than \u003cem\u003eMet\u003c/em\u003e-TR61 on the 3rd, 5th, and 7th days after infection when death numbers were recorded (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Only 43.6% of the cadavers were found to have mycosis for commercial products (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Under the same conditions and time, the blank product genetated 19.6% of \u003cem\u003eM. persicae\u003c/em\u003e (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Mortality rates in the control group were 17.6% of \u003cem\u003eM. persicae\u003c/em\u003e after the 7th-day post-treatment (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSusceptibility of \u003cem\u003eMyzus persicae\u003c/em\u003e nymphs exposed at 1 \u0026times; 10\u003csup\u003e8\u003c/sup\u003e conidia/ml of \u003cem\u003eMet\u003c/em\u003e-TR61 in leaf disc and pot experiment under laboratory conditions.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"9\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eCharacters\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"4\" nameend=\"c5\" namest=\"c2\"\u003e \u003cp\u003eThe leaf disc experiments\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"4\" nameend=\"c9\" namest=\"c6\"\u003e \u003cp\u003eThe pot experiments\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMortality% \u0026plusmn;SE\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCI 95%\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMycosis %\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eCI 95%\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eMortality% \u0026plusmn;SE\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eCI 95%\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eMycosis %\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eCI 95%\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eMet\u003c/em\u003e-TR61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e78.33\u0026thinsp;\u0026plusmn;\u0026thinsp;1.15 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e73.36\u0026ndash;83.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e92.66\u0026thinsp;\u0026plusmn;\u0026thinsp;1.27 \u003csup\u003ex\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e89.03-110.96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e79.00\u0026thinsp;\u0026plusmn;\u0026thinsp;1.52\u003csup\u003eA\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e72.42\u0026ndash;85.57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e81.33\u0026thinsp;\u0026plusmn;\u0026thinsp;1.33 \u003csup\u003em\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e75.59\u0026ndash;87.07\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMET52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e75.00\u0026thinsp;\u0026plusmn;\u0026thinsp;2.88\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e62.57\u0026ndash;87.42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e43.66\u0026thinsp;\u0026plusmn;\u0026thinsp;1.56 \u003csup\u003ey\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e35.54\u0026ndash;61.92\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e72.33\u0026thinsp;\u0026plusmn;\u0026thinsp;1.52 \u003csup\u003eB\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e65.75\u0026ndash;78.90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e76.33\u0026thinsp;\u0026plusmn;\u0026thinsp;1.15 \u003csup\u003en\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e71.03\u0026ndash;80.96\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBlank product\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e19.69\u0026thinsp;\u0026plusmn;\u0026thinsp;1.15 \u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e14.72\u0026ndash;24.65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e16.33\u0026thinsp;\u0026plusmn;\u0026thinsp;1.15 \u003csup\u003eC\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e11.36\u0026ndash;21.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eNG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e18.32\u0026thinsp;\u0026plusmn;\u0026thinsp;1.76 \u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e10.73\u0026ndash;25.91\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e10.66\u0026thinsp;\u0026plusmn;\u0026thinsp;0.66 \u003csup\u003eD\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e7.79\u0026ndash;13.53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eNG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"9\"\u003eNote: Mortality% \u0026plusmn; SE within a column followed by the upper case letter indicates the differences between mortality and lower case indicates the difference in the rate of mycosis after mortality (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) Tukey\u0026rsquo;s LSD test, P\u0026thinsp;\u0026le;\u0026thinsp;0.05). NS: Nonsignificant, NG: Non-growth fungus\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe pot experiment results revealed that plant vitality and flowering status were high and aphid populations were low in the seedlings where the \u003cem\u003eMet\u003c/em\u003e-TR61 was applied (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). On the contrary, plant viability and flowering status were incredibly low in the control group and the blank product, the seedlings died, and the number of aphids increased gradually in the first 3 days post-infection. \u003cem\u003eMet\u003c/em\u003e-TR61 was determined as the most effective oil-based formulation in the pots experiment with 79% mortality (F \u003csub\u003e(3,8)\u003c/sub\u003e)\u0026thinsp;=\u0026thinsp;807.51, df\u0026thinsp;=\u0026thinsp;3, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). As a positive control, while MET52 caused 72,33% insecticidal effects on the pest, the blank product showed 16.3% mortality (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). It was determined that commercial products on \u003cem\u003eM. persicae\u003c/em\u003e nymphs were similar to each other in terms of mortality (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Mean aphid mortality in control was determined at 10.6% (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). No fungal infection and mycosis were detected on aphids used in the blank product and control (p\u0026thinsp;\u0026lt;\u0026thinsp;0,05) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eEffect of\u003c/b\u003e \u003cb\u003eMet\u003c/b\u003e\u003cb\u003e-TR on different aphid species\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003cem\u003eMet\u003c/em\u003e-TR also produced remarkably high mortality values on other aphid species used in the study (F \u003csub\u003e(3,8)\u003c/sub\u003e)\u0026thinsp;=\u0026thinsp;1.28, df\u0026thinsp;=\u0026thinsp;3, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Mortality rates were calculated as 79% on \u003cem\u003eA. fabae\u003c/em\u003e, 84% on \u003cem\u003eB. brassicae\u003c/em\u003e and 82% on \u003cem\u003eM. rosae\u003c/em\u003e with 10\u003csup\u003e8\u003c/sup\u003e conidia/ml concentration on the 7th-day post-infection (F \u003csub\u003e(3,11)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;4.003, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and mycosis rates of the cadavers were found as 84.0, 90.6 and 85.0%, respectively. The mortality values of the commercial product (MET52) on the same aphid species were determined to be 69% and 75% (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Also, the blank product was between 18% and 20% under the same conditions and time (p\u0026thinsp;\u0026lt;\u0026thinsp;0.059 (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSusceptibility of different aphid nymphs exposed at 1 \u0026times; 10\u003csup\u003e8\u003c/sup\u003e conidia ml\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of \u003cem\u003eMet\u003c/em\u003e-TR61\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eCharacters\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e\u003cem\u003eBrevicoryne brassicae\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e\u003cem\u003eAphis fabae\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e\u003cem\u003eMacrosiphum rosae\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMortality% \u0026plusmn;SE\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCI 95%\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMortality% \u0026plusmn;SE\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eCI 95%\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eMortality% \u0026plusmn;SE\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eCI 95%\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eMet\u003c/em\u003e-TR61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e84.00\u0026thinsp;\u0026plusmn;\u0026thinsp;1.15 \u003csup\u003eA\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e79.03\u0026ndash;88.96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e79.00\u0026thinsp;\u0026plusmn;\u0026thinsp;1.73\u003csup\u003eB\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e71.54\u0026ndash;86.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e82.00\u0026thinsp;\u0026plusmn;\u0026thinsp;1.15 \u003csup\u003eA\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e77.03\u0026ndash;86.96\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMET52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e73.33\u0026thinsp;\u0026plusmn;\u0026thinsp;1.76\u003csup\u003eC\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e65.74\u0026ndash;80.92\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e69.33\u0026thinsp;\u0026plusmn;\u0026thinsp;2.60 \u003csup\u003eD\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e58.13\u0026ndash;80.53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e71.00\u0026thinsp;\u0026plusmn;\u0026thinsp;1.15\u003csup\u003eCD\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e66.03\u0026ndash;75.96\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBlank product\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e18.33\u0026thinsp;\u0026plusmn;\u0026thinsp;2.02\u003csup\u003eE\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e9.60\u0026thinsp;\u0026plusmn;\u0026thinsp;27.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e18.00\u0026thinsp;\u0026plusmn;\u0026thinsp;1.73\u003csup\u003eE\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e10.54\u0026ndash;25.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e18.33\u0026thinsp;\u0026plusmn;\u0026thinsp;2.02\u003csup\u003eE\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e9.60\u0026thinsp;\u0026plusmn;\u0026thinsp;27.05\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e11.33\u0026thinsp;\u0026plusmn;\u0026thinsp;1.76\u003csup\u003eF\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.74\u0026thinsp;\u0026plusmn;\u0026thinsp;18.92\u003csup\u003eH\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8.33\u0026thinsp;\u0026plusmn;\u0026thinsp;1.45\u003csup\u003eG\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e2.08\u0026ndash;14.58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e9.33\u0026thinsp;\u0026plusmn;\u0026thinsp;1.45 \u003csup\u003eG\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e3.08\u0026thinsp;\u0026plusmn;\u0026thinsp;15.58\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003eNote: Mortality% \u0026plusmn; SE within a column followed by the letter indicates the differences in mortality (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) Tukey\u0026rsquo;s LSD test, P\u0026thinsp;\u0026le;\u0026thinsp;0.05.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn particular, the effect of the new product on \u003cem\u003eB. brassicae\u003c/em\u003e was found to be higher and significantly different than \u003cem\u003eA. fabae\u003c/em\u003e (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). It was determined that the results on \u003cem\u003eB. brassicae\u003c/em\u003e and \u003cem\u003eM. rosea\u003c/em\u003e were close and there was no significant difference (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). When we compared the effect of commercial and blank products on aphids, it was determined that there was a similar mortality rate in all insects and no significant difference between them, but a significant difference in the control group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Moreover, the aphid populations increased rapidly in the control and the blank product applications.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe main objective of biocontrol research is to develop novel, useful, and readily applicable mycoformulations for use against target pests. The main goal of this study is to create a new oil-based mycoinsecticide from a strain of \u003cem\u003eMetarhizium anisopliae\u003c/em\u003e that is native to T\u0026uuml;rkiye to control the \u003cem\u003eMyzus persicae\u003c/em\u003e aphid species as well as a few other aphid species.\u003c/p\u003e \u003cp\u003eFungi such as \u003cem\u003eLecanicillium\u003c/em\u003e spp., \u003cem\u003eBeauveria bassiana\u003c/em\u003e, \u003cem\u003eMetarhizium anisopliae\u003c/em\u003e, and \u003cem\u003eIsaria\u003c/em\u003e spp. are entomopathogenic fungi that play significant roles in regulating insect populations. Many entomopathogenic fungi that were effective against various insects in our country and all over the world were isolated from insect and soil samples, characterized according to their molecular and morphological features and their potential for use in the biological control of agricultural pests was determined (Biryol et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Eski et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Kocacevik et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; 2016; Gurulingappa et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). \u003cem\u003eM. anisopliae\u003c/em\u003e is the second most widely exploited entomopathogenic fungi in biocontrol studies next to \u003cem\u003eB. bassiana\u003c/em\u003e, and it is known to attack more than 200 insect species belonging to different orders (Jitendra et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Chen et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Entomopathogenic hyphomycetes-based mycoinsecticides have been tested against sucking insects such as aphids in recent years, and their effectiveness against such pests has been demonstrated (Milner, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e1997\u003c/span\u003e; Vandenberg et al. \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). The global demand for biopesticides is also increasing day by day. Most of these mycoinsecticides are produced in America, Europe, and Asia (Faria and Wraight \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). The mycoinsecticide AFIDISIDAL-OD \u003cem\u003eMet\u003c/em\u003e-TR61 based on \u003cem\u003eM. anisopliae\u003c/em\u003e developed in this study was produced using solid-substrate fermentation. Spore yield can also be increased with carbon and nitrogen sources such as rice, yeast extract, corn maceration liquid, or molasses, which are used as substrates or carriers (Talwar \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). This method was preferred especially because of the high spore yield and some problems encountered in the production of strains of this species in a solid substrate. Spores obtained from production on a solid substrate can be used in different types of formulations, such as granular and spray (aqueous and oil-based) (Leland \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). EPF-based biopesticides tend to be more effective when formulated in oil, as the conidia are evenly distributed on the insect cuticle and leaf surface. In contrast, aqueous spore formulations tend to remain as drops on the surface after application, which may reduce their efficacy (Inyang et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Wraight et al. \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). In bioassay studies, we determined that AFIDISIDAL-OD \u003cem\u003eMet-\u003c/em\u003eTR61 remained active in host plants after application. The benefits of oil-based formulations for pest control, such as mortality and protection against adverse environmental effects, have been widely reported (Hedimbi et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Lopes et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Oliveira et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). It was determined that the aphids we used in the current study were sensitive to both the new product (AFIDISIDAL-OD \u003cem\u003eMet\u003c/em\u003e-TR61) and the commercial product. \u003cem\u003eMet\u003c/em\u003e-TR61 caused higher mortality than the commercial biopesticide products in our bioassay on the four aphid species. Oil-based carrier fungus conidia are more resilient to environmental stress factors, making them more resilient to environmental stress factors such as humidity, temperature, and UV radiation, according to research by Kaaya and Hassan (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2000\u003c/span\u003e) and Batta (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). This may extend the conidia's shelf life and vitality and raise the fungus's likelihood of spreading infections. In a study, Biryol et al. (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) reported that the isolate used in the current study was resistant to environmental factors (UV-B and temperature) experiments and had high virulence. The results of the mortality experiments strongly support the development of biopesticides for the control of the four aphid species. In the study conducted by Mohammed and Hatcher (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) on the 3rd stage \u003cem\u003eM. persicae\u003c/em\u003e nymphs, it was determined that Mycotal caused 100% mortality with 1 \u0026times; 10\u003csup\u003e10\u003c/sup\u003e conidia/ml concentration in laboratory conditions on the 9th day. In the current study, \u003cem\u003eMet\u003c/em\u003e-TR61 yielded a mortality rate of 79% in 7th days in a pot experiment of 1 \u0026times; 108 conidia /ml concentration. The study by Lopes and Faria (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) assessed the effect of oil-based formulation on the coffee beetle species \u003cem\u003eHypothenemus hampei\u003c/em\u003e. According to the findings of the study, the oil formulation had a 20% more considerable mortality rate after six days than the pure conidia suspension at a concentration of 2 x 10\u003csup\u003e7\u003c/sup\u003e conidia/ml. According to the research findings, unformulated spore suspensions prepared in water, particularly oil-based formulations, are more effective when compared to the results of both laboratory and field applications of pests, and the reason for this effect is the spread and adhesion of the conidia on the hydrophobic cuticle of the host arthropod (David-Henriet et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e1998\u003c/span\u003e). It has even been included in bioassays to enhance the mortality rate of \u003cem\u003eSpodoptera litura\u003c/em\u003e and \u003cem\u003eHelicoverpa armigera\u003c/em\u003e (Vimala Devi and Prasad \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; Vimala Devi and Hari \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). According to Albernaz et al. (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2009\u003c/span\u003e), M. \u003cem\u003eanisopliae\u003c/em\u003e formulations based on 10% sunflower oil showed increased ovicidal activity against \u003cem\u003eAedes aegypti\u003c/em\u003e. The oil has no impact on germination because of its low volatility and compatibility with environmental conditions (ULV). It has been demonstrated to improve \u003cem\u003eM. anisopliae\u003c/em\u003e conidia efficacy (Albernaz et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Bateman et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Batta \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2003\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eEPF-based formulations must be effective before the immature stages of these pests reach the reproductive age, which means that they are effective in a short time. The maturation period of \u003cem\u003eM. persicae\u003c/em\u003e is 7.6 days, according to a study (Hong et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), and when we transplanted it into our study, it was determined that the new mycopesticide was effective on this pest from the second day, and the pest population decreased.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003ePrevious research on \u003cem\u003eMetarhizium anisopliae\u003c/em\u003e KTU-51, including isolation, identification, and insecticidal activity studies, has revealed that KTU-51 holds great promise for biological pest control. The results of this study indicate that the local mycoinsecticide developed from this isolate is also effective against other aphids, particularly \u003cem\u003eM. persicae\u003c/em\u003e, and can be used successfully for the biological control of these important agricultural pests. Studies on the commercialization of an effective, environmentally friendly, and ecologically compatible AFIDISIDAL-OD \u003cem\u003eMet-\u003c/em\u003eTR61 must be conducted as soon as possible.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors deeply appreciate the financial support of KTU-BAP (The Scientific Research Committee of Karadeniz Technical University) for the project FDK-2017-5896.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study was designed by ID and SB, with SB carrying out the research. ID and SB contributed to the initial draft of the manuscript, with both authors involved in subsequent review and editing. All authors have read and approved the final version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Karadeniz Technical University KTU-BAP (The Scientific Research Committee) has funded this research for the project FDK-2017-5896.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated and analyzed during this study are indicated in the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAbbott WS (1925) A method of computing the effectiveness of an insecticide. 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Biol Control 97:31\u0026ndash;47. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.biocontrol.2016.02.016\u003c/span\u003e\u003cspan address=\"10.1016/j.biocontrol.2016.02.016\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"international-journal-of-tropical-insect-science","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jtis","sideBox":"Learn more about [International Journal of Tropical Insect Science](http://link.springer.com/journal/42690)","snPcode":"42690","submissionUrl":"https://www.editorialmanager.com/jtis/default2.aspx","title":"International Journal of Tropical Insect Science","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Aphid, biocontrol, mycoinsecticide, oil-based formulation, solid-state fermentation","lastPublishedDoi":"10.21203/rs.3.rs-3314382/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3314382/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe research aims to propose an appropriate and efficient mycoinsecticide from a local \u003cem\u003eMetarhizium anisopliae\u003c/em\u003e (Ascomycota: Hypocreales) strain (KTU-51) against several aphid species (Hemiptera: Aphididae; \u003cem\u003eMyzus persicae\u003c/em\u003e Sulz., \u003cem\u003eAphis fabae\u003c/em\u003e Scop., \u003cem\u003eBrevicoryne brassicae\u003c/em\u003e L. and \u003cem\u003eMacrosiphum rosae\u003c/em\u003e L.), which are the most important agricultural pests. \u003cem\u003eM. anisopliae\u003c/em\u003e produced spores in large quantities utilizing solid-state fermentation (SSF) with rice as a substrate. An oil-based mycoinsecticide called AFIDISIDAL-OD Met-TR61 was improved by incorporating spores harvested from the sporulated biomass. The product provided a more deathful effect than commercial products against all the aphis species under laboratory conditions. \u003cem\u003eMet\u003c/em\u003e-TR61 with 10\u003csup\u003e8\u003c/sup\u003e spores/ml concentration yielded 78.3% mortality in the leaf disc experiment and 79% in the pot experiment on \u003cem\u003eMyzus persicae\u003c/em\u003e. Other aphids were also found to be extremely sensitive to the product. In this particular study, an oil-based mycoinsecticide was developed and its efficacy for biological control of aphid species was assessed. The study\u0026rsquo;s findings indicate that the mycoinsecticide has the potential to be a workable and effective alternative to conventional chemical insecticides for controlling aphid populations.\u003c/p\u003e","manuscriptTitle":"A mycoinsecticide from Metarhizium anisopliae (Ascomycota: Hypocreales) based on a solid-state fermentation method against some aphid species in Türkiye","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-14 18:10:23","doi":"10.21203/rs.3.rs-3314382/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2024-03-12T19:19:20+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-03-12T18:57:33+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"International Journal of Tropical Insect Science","date":"2023-09-05T13:37:42+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2023-09-04T14:51:26+00:00","index":"","fulltext":""},{"type":"submitted","content":"International Journal of Tropical Insect Science","date":"2023-08-31T12:20:11+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"international-journal-of-tropical-insect-science","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jtis","sideBox":"Learn more about [International Journal of Tropical Insect Science](http://link.springer.com/journal/42690)","snPcode":"42690","submissionUrl":"https://www.editorialmanager.com/jtis/default2.aspx","title":"International Journal of Tropical Insect Science","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"b630cdc7-63fb-4e7f-8590-dc82cfc8b787","owner":[],"postedDate":"March 14th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-04-13T16:07:55+00:00","versionOfRecord":{"articleIdentity":"rs-3314382","link":"https://doi.org/10.1007/s42690-026-01814-6","journal":{"identity":"international-journal-of-tropical-insect-science","isVorOnly":false,"title":"International Journal of Tropical Insect Science"},"publishedOn":"2026-04-08 15:58:24","publishedOnDateReadable":"April 8th, 2026"},"versionCreatedAt":"2024-03-14 18:10:23","video":"","vorDoi":"10.1007/s42690-026-01814-6","vorDoiUrl":"https://doi.org/10.1007/s42690-026-01814-6","workflowStages":[]},"version":"v1","identity":"rs-3314382","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3314382","identity":"rs-3314382","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}