Compatibility and synergistic interactions of fungi, Metarhizium anisopliae, and insecticide combinations against the cotton aphid, Aphis gossypii Glover (Hemiptera: Aphididae)

Aphids are major pests affecting cereals, vegetables, fruit, forestry and horticultural produce. A multimodal approach may be an effective route to controlling this prolific pest. We assessed the individual and combined effect of eight insecticides and the entomopathogenic fungi, Metarhizium anisopliae (Metschin.) against the cotton aphid, Aphis gossypii Glover (Hemiptera: Aphididae), under laboratory conditions. Six of the insecticides tested were found to be highly compatible (flonicamid, imidacloprid, nitenpyram, dinotefuran, pyriproxyfen and spirotetramat), showing positive integration with the fungus and were selected for bioassays. The combination mixtures (1:1 ratio of M. anisopliae: insecticide) were significantly more toxic to A. gossypii than individual treatments. Maximum mortality (91.68%) of A. gossypii was recorded with combination of flonicamid and M. anisopliae (2.4 × 106 cfu/ml) 72 h after application. While minimum mortality (17.08%) was observed with the individual treatment of M. anisopliae (2.4 × 106 cfu/ml). The insecticides revealed toxicity consistent with their compatibility with M. anisopliae, ranking for efficacy exactly as they did for compatibility. In addition, the synergy factor (SF) and co-toxicity coefficient (CTC) values indicated synergistic interactions at different time intervals. The synergistic efficacy revealed the potential of fungus-insecticide integration against sucking insect pests.

www.nature.com/scientificreports/ Aphids are small sap-sucking insects. Among the 5000 described species, 450 aphid species cause intense damage to crop and ornamental plants around the world 1 . They are distributed globally but most commonly found in temperate zones where species diversity is also much higher compared to the tropics 2 . Aphids are considered serious pests because they reach a high population density and can develop resistance to insecticides in a short period of time 3,4 . The cotton aphid, Aphis gossypii Glover (Hemiptera: Aphididae), is a highly polyphagous pest. It causes serious damage like leaf curling, leaf deformation and transmits at least 76 viral diseases including potyvirus, cucumber mosaic virus and zucchini yellow virus to a wide range of crops 5 . Aphid nymphs and adults deplete photo assimilates through their feeding and devitalize the plant in the process 6 ). Aphids also secrete honeydew which attracts black sooty mould that stains cotton fiber and blocks photosynthesis. The honeydew also causes sticky cotton during mechanical harvesting, ginning, and processing 7 . Several control measures including host plant resistance, cultural, biological and chemical control are utilized to keep the pest population below economic injury level 8 . Sucking insect pests like aphids and whiteflies can be controlled by using neonicotinoids 9 . Neonicotinoids act as inhibitor on nicotinic acetylcholine receptors in the central nervous system 10 . The intensive use of insecticides to control cotton aphids has led to populations that are now resistant to several classes of insecticides 11 . In addition, pesticides can cause serious problems of environmental contamination and adverse effects on beneficial insects such as bee populations [12][13][14] . Biopesticides offer a route to protecting the crop while reducing the reliance on synthetic insecticides 15 . Entomopathogenic fungi (EPF) have been found to be effective as a biopesticide 16 and have potential to minimize the target pest populations on multiple crops [17][18][19][20] . Moreover, 750 species of EPF are known to inoculate insect pests 21 . One commonly used entomopathogenic fungi is Metarhizium anisopliae (Metschin.), which has been shown to be effective for control against 200 insect species 22 including Aphis gossypii 23,24 ). More than 150 insect biocontrol products based on fungal entomopathogens have been commercialized with over 75% of these products based on the hypocrealean fungi M. anisopliae, Beauveria bassiana, Isaria fumosorosea, and B. brongniartii 16 , however this number is expected to have increased since the last major market evaluations were conducted. Entomopathogenic fungi are generally considered slow-acting, taking longer than conventional methods to achieve sufficient insect mortality. The technique of combining EPF into a management strategy with faster-acting materials may be the solution to this problem. The synergistic action of mycoinsecticides with chemical insecticides can increase mortality and reduce the time until death in insects [25][26][27][28] . The combined use of fungal pathogens and the full, or reduced, dose of chemical insecticides is a promising pest-control option. The application of synergists can effectively enhance the cost-effectiveness and eco-friendliness of insecticides by reducing the required quantity and extending the residual activity. By attacking the pest through a different mode of action, they are equally important as an alternative for resistance management. The data is lacking regarding the compatibility of EPF with insecticides and synthetic insecticide combinations with mycoinsecticides are rarely evaluated against aphids. In this study we gauge the compatibility of different insecticides with M. ansopliae and assess their toxicity to a prominent aphid pest.

Materials and methods
Metarhizium anisopliae culture. Potato Dextrose Broth (PDB) media was used 56 in a 1000 ml Erlenmeyer flask and autoclaved at 121 °C for 20 min as previously described 29 . A disc of the cultured fungi approximately 5 mm in diameter was taken from its Petri dish and added into the prepared media under a laminar air flow chamber and kept at 25 ± 1 °C for 5 days before being transferred to a shaking incubator (Firstek Scientific, Tokyo, Japan) at 180 rpm for 48 h at 28 ± 1 °C. An optical density of 0.5 was measured with an OD meter (BIOLOG MODEL-21907; BIOLOG INC.) at λ 600 nm. This was achieved by dilution to maintain uniform conidia density (10 6 CFU mL -1 ) prior to application. Inoculum and saline buffer (0.85% NaCl w/v) at ratios 1:9 and 2:18 were mixed to prepare M. anisopliae suspensions containing 10 6 CFU mL −1 . To achieve these populations, OD 0.4 and 0.3 samples were adjusted prior to application.
Insecticides compatibility with M. anisopliae. To assess compatibility, the effect of different insecticides (flonicamid, imidacloprid, nitenpyram, dinotefuran, pymetrozine, pyriproxyfen, spirotetramat and matrine) on the radial growth of M. anisopliae was evaluated. The recommended field doses of insecticides were added to potato dextrose agar (PDA) in an Erlenmeyer flask before solidification. After mixing thoroughly, the media was transferred to Petri dishes and with gentle shaking allowed to solidify. Using a micropipette, M. anisopliae formulation (2.4 × 10 6 CFU mL -1 ) was inoculated in each petri dish on media. The Petri dishes were sealed and placed in an incubator maintained at 25 ± 1 °C, 80 ± 5% relative humidity. The media without insecticide (Tween 80, 0.05%) was used as a control treatment. Fungal colony diameter was calculated after 3 days of inoculation using Vernier calipers. Treatment groups were compared to growth observed in the control to evaluate the potential impact of the insecticide on colony development. Determination of synergistic effect. The toxicity of combined and isolated treatments was calculated based on LC 50 and LC 90 of insecticides and combination treatments with EPF using probit analysis. The cotoxicity coefficient 30 and synergy factor 31 for mixed formulation were calculated utilizing the LC 50 and LC 90 identified for each treatment.
Within this system, a SF value > 1 indicates synergism and an SF value < 1 indicates antagonism 32,33 . Statistical analysis. Percentage mortality of aphids was calculated by Abbot's Formula 34 . The experiment was carried out under controlled condition inside the incubator (POL-EKO_APARA TUR A SP.J. S02ADF 180665) and collected data were checked for normality and homogeneity of variance using Shapiro-Wilk test. The P value obtained was larger than probability value of 5% which indicated that distribution of data was normal. Mortality data were recorded daily after treatment and analyzed using the Statistix software version 8.1. Percentage corrected mortality data were analyzed by main effects one way ANOVA through Multivariate General Linear Model (MGLM) Technique 35 , using a STATISTICA software version 10.0 to determine the parameters of significance and mean values for different treatments and followed by a Tukey's honestly significant difference (HSD) test with significant differences recognized when p < 0.05 36 . The LC 50 , LC 90 , chi-square and confidence interval values for each extract were also calculated by Probit analysis using the Minitab Statistical Program 37 . Regression between aphid's mortality and concentrations of insecticides was also established, using linear regression and Pearson correlation analysis at 5% level of probability. Scattered diagrams for concentration of each insecticides (alone or in combination) and mortality of aphid were also drawn to construct fitted simple regression line of mortality on concentrations.

In vitro study on compatibility of insecticides with M. anisopliae. Effects of the insecticides on
M. anisopliae vegetative growth showed that all tested formulations significantly inhibited the fungal growth. However, insecticides did not all inhibit M. anisopliae growth to the same extent. The greatest radial growth of the fungi with any insecticide treatment was observed with flonicamid with a colony diameter of 4.74 mm at the lowest concentration. The mean diameters of colonies based on 3 replicates were 4.65, 4.37, 3.96, 3.79, and 3.69 mm for imidacloprid, nitenpyram, dinotefuran, pyriproxyfen, and spirotetramat respectively. The pymetrozine and matrine treatments led to the lowest radial growth (Fig. 1).
Co-toxicity coefficient = Toxicity of insecticide (alone) Toxicity of insecticide with fungal extract × 100 Synergy factor (SF) = Toxicity of insecticide (alone) Toxicity of insecticide with fungal extract   (Table 3).
For dinotefuran, it was found that a combination with the EPF resulted in a synergistic interaction in all samples except for the LC 90 at 24 h where antagonism was observed (SF = 0.754).
Pyriproxyfen showed synergistic interactions with M. anisopliae at all levels of data analysis ( Table 4). The  (Table 4). For evaluation using the LC 50 , synergistic interactions were observed for all time points (SF > 1).

Discussion
Insecticides have the potential to affect the various developmental stages of entomopathogenic fungi. The effect of an insecticide on conidial germination is the most important factor in determining fungus-insecticide compatibility 38,39 . We found that the insecticides tested did reduce vegetative growth and sporulation compared The six insecticides with better compatibility (great colony growth) were chosen for toxicity bioassays. Letters above the bars indicate differences between treatments as determined by ANOVA followed by Tukey HSD.
Those not sharing a letter are significantly different (p < 0.05). www.nature.com/scientificreports/ to the control but not always to the extent that would preclude compatibility of the insecticides tested, flonicamid, imidacloprid, nitenpyram, dinotefuran, pyriproxyfen, and spirotetramat exhibited good compatibility with M. anisopliae. Significantly reduced fungal colony diameter was observed for pymetrozine and matrine treatments. The insecticides caused different levels of inhibition of germination, vegetative growth, and sporulation of M. anisopliae. This is dependent on compounds present that block conidia metabolic functions as well as concentrations of the active compounds 40,41 . Oliveira 42 reported that, molecules analogous to prosthetic groups diffuse to the cytoplasm where they bind to specific receivers affecting membrane permeability and enzymatic synthesis, consequently affecting metabolic processes. The same mechanism of inhibition is likely to be responsible for conidial germination and vegetative growth differences in M. anisopliae. M. anisopliae have been employed effectively to control several insect pest species, including other aphid species such as Lipaphis erysimi 43 . Variation in interaction modalities (synergistic, antagonistic or neutral) of EPF  T1  T2  T3  T4  T5  T6  T7  T8  T9  T10  T11  T12  T13  T14  Mortality (%age)   Treatments   C 1 C2 C 3   a   ab   ab  abc abc  bcd  bcd  cde  de  de  de  ef  f   a  ab  ab  b  d  c  b  c  b  bcd  cde  cde  de  e   a  a  ab  abc  cd  cde  def  def  ef  f  f  ef   ef   bc   0   20   40   60   80   100   T1  T2  T3  T4  T5  T6  T7  T8  T9  T10  T11  T12 T13 T14  T1  T2  T3  T4  T5  T6  T7  T8  T9  T10  T11  T12  T13 50,51 .

Treatments
Looking at the mustard aphid, Lipaphis erysimi, Purwar and Sachan 52 also observed enhanced efficiency through an insecticide-EPF combination.   (Tables 2, 3, 4) The antagonistic effect observed for imdiacloprid, dinotefuran, and spriotetramat at 24 h post exposure may be related to issues of compatibility, particularly suppression of EPF activity before the colony fully establishes, especially given that this antagonism is not observed at later time points. Ultimately, the combined treatments proved to be more effective than individual applications of all compounds tested (insecticides and M. anisopliae). The high values of co-toxicity coefficients, which were accompanied by insect mortalities > 90% for some treatments, illustrate the effectiveness of this dual-attack method of insect pest control. This finding is supported by previous studies, such as Quintela and McCoy 53,54 which found that B. bassiana and M. anisopliae combined with sublethal doses of imidacloprid as a contact or oral treatment increased the mortality synergistically in the weevil, Diaprepes abbreviatus. Or the additive effect that has been observed with aphid species when B. bassiana is combined with a botanical pesticide, showing efficacy enhanced even in lower concentrations 55 .
From our findings we propose that dual modality approach is highly effective in achieving pest mortality. However, given the parity of compatibility of the insecticide with the EPF and its efficacy as a combined treatment, we identify that the insecticide's direct effect on the EPF may be the primary criterion deciding success of a combination treatment.

Conclusion
The combination of M. anisopliae with insecticides showed a synergistic effect and led to higher mortality of the cotton aphid, A. gossypii. If laboratory evidence for synergistic effects of M. anisopliae and insecticides against A. gossypii applies under greenhouse or field conditions, this control solution could mitigate potential issues related to environmental contamination, non-target impacts and pesticide resistance. However, further studies on the mechanism of toxicity of these combinations are needed. www.nature.com/scientificreports/ www.nature.com/scientificreports/

Data availability
The data used and analyzed during this project are available from the corresponding author on reasonable request. www.nature.com/scientificreports/