Source of maize grains
The variety of maize used during all experimentation was Shaba provided by IRAD, Wakwa station in the Adamawa region of Cameroon. Before experimentation, broken grains, the pieces of stone, sand and other foreign materials were removed from the stock. Then, the maize was kept in the freezer at − 20 °C for 14 days to allow its disinfestations. After disinfestations from all types of living organisms, the maize was kept in ambient conditions of laboratory for 14 days to allow its acclimatisation. After all these steps, the maize was ready for use as substrate for insect rearing and bioassays.
Insect rearing
Adults of S. zeamais were obtained from a colony maintained in rearing since 2005 in the Applied Chemistry Laboratory of the University of Ngaoundere. Then, the insect culture was transferred and kept in Crop Protection Laboratory of IRAD Bambui, North-West region of Cameroon. The weevils were reared on disinfested maize in 900-mL glass jars and kept under laboratory conditions (23.08 ± 2.05 °C and 74.67 ± 14.36% r.h.). The culture was maintained and used as source of S. zeamais for bioassays.
Preparation of Hymenocardia acida wood ash
Stems and branches of H. acida were collected in Ngaoundéré, Adamawa region of Cameroon (latitude 7°25ʹ North and longitude 13°35ʹ East, altitude of 1151 m above sea level). The identity of the plants was confirmed at the Cameroon National Herbarium in Yaounde, where voucher samples were deposited. H. acida is registered on number 50114/HNC. Woods were air-dried until moisture was completely lost and burnt separately in a traditional kitchen normally used in the region. The obtained ash was sieved and packaged in glass jars, labelled and kept in a freezer (at − 4 °C) until subsequent use in the bioassays.
Preparation of Plectranthus glandulosus leaf powder
Leaves of P. glandulosus were collected in July 2012 in Ngaoundere, head quarter of the Adamawa region of Cameroon (latitude 7°25ʹ North and longitude 13°35ʹ East, altitude of 1151 m above sea level). The identity of the plant was confirmed at the Cameroon National Herbarium in Yaounde on number 7656/SRF. The leaves were dried at room temperature for 7 days and then crushed. The crushed leaves were ground until the powder passed through a 0.20-mm sieve. Then, a part of powder was stored in a freezer at − 20 °C until needed for bioassays and the other part was used for essential oil extraction.
Preparation of binary combinations
The products were mixed in the following proportions to constitute the different binary combinations:
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25% P. glandulosus leaf powder and 75% H. acida wood ash: 25PG75HA;
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50% P. glandulosus leaf powder and 50% H. acida wood ash: 50PG50HA;
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75% P. glandulosus leaf powder and 25% H. acida wood ash: 75PG25HA.
Analysis of volatile compounds of Plectranthus glandulosus leaf powder
The essential oil was extracted by hydrodistillation during 4 h using a Clevenger-type apparatus. The extracted oil was kept in a brown bottle at 4 °C to avoid degradation of chemical compounds by light until needed for gas chromatography–mass spectrometry (GC–MS) analysis.
Gas chromatography–mass spectrometry analysis was carried out with a chromatograph, model Agilent 7890A GC, equipped with an automatic injector and a column HP-1MS (15 m × 0.25 mm d.i; 0.25 µm film thickness) coupled to a mass detector Agilent 7890A MSD. The molecules were bombarded by an electronic beam of 70 eV. The gaze vector was helium (1 mL/min) with a pressure of 25 psi at the beginning of the column. The injector temperature was 250 °C. The programming of temperature consisted of a rise from 60 to 230 °C with the range of 2 °C/min and then 35 °C/min to reach 230 °C. The injection was done by split mode with the coefficient of 1/180. The injected quantity of essential oil of P. glandulosus was 0.2 µL. The detection was done by a quadripolar analyser constituted by an assembling of four parallel cylindrical electrodes. The bombing of essential oil by the electronic beam of 70 eV induced its ionisation and its fragmentation. Then, the positive ionic fragments formed the characteristic mass spectrum of compounds. The obtained spectra were compared with computerised database using NIST/EPA/NIH Mass Spectral Library, Wiley Register of Mass Spectral Data [23] and König et al. [24].
Determination of Hymenocardia acida wood ash mineral contents
The sample of ash was calcinated at 450 °C for 24 h using incinerator for a complete mineralisation [25]. Calcinated ash was dissolved in nitric acid (HNO3) 1 M for digestion and then boiled. The solution was filtered after cooling. The filtrate obtained was used to proportion the following minerals: P, K, Ca, Mg, Na, Fe, Mn, Zn and Pb. Ca, K and Na were proportioned by flame photometry, while Mg, Fe, Mn, Zn and Pb were proportioned by atomic absorption spectrometry. The content of phosphate was measured by molecular absorption spectrophotometry.
Toxicity bioassay
The toxicity bioassay was carried out under ambient conditions of the laboratory. During experimentation, the temperature and relative humidity were recorded using a data logger (Data logger Model EL-USB-2, LASCAR, China). Four concentrations from each combination were considered. The masses of 0.25, 0.5, 1 and 2 g of P. glandulosus leaf powder and H. acida wood ash as well as their binary combinations were separately added to 50 g of maize in glass jars to constitute the contents of 5, 10, 20 and 40 g/kg, respectively. The insecticidal materials plus grain were thoroughly mixed by manual shaking. The controls consisted of substrate without insecticidal products. A group of 20 insects of mixed sexes and 7- to 14-days-old were added into each jar containing the treated or untreated grains. All treatments were replicated four times, and the experiment was arranged in a completely randomised design. Mortality was recorded 1, 3, 7 and 14 days post-infestation.
The co-toxicity coefficient per P. glandulosus leaf powder–H. acida wood ash mixture was calculated: A co-toxicity coefficient of less than 80 is considered as antagonistic, between 80 and 120 as additive and higher than 120 as synergistic [26]. When mixture (M) compounds of two parts (A and B) and both components have LC50, then the following formulae are used (A serving as standard, it is represented in this study by P. glandulosus leaf powder, B represents wood ash for H. acida):
$${\text{Toxicity}}\; {\text{index}}\, \left( {\text{TI}} \right)\;{\text{of}}\; A = 100,$$
$${\text{Toxicity}}\; {\text{index}}\, \left( {\text{TI}} \right)\;{\text{of}}\; B = \frac{{{\text{LC}}_{50} \;{\text{of}}\; A}}{{{\text{LC}}_{50} \;{\text{of}}\; B}} \times 100,$$
$${\text{Actual}} \;{\text{TI}}\; {\text{of}} \;M = \frac{{{\text{LC}}_{50 } \;{\text{of}}\; A}}{{{\text{LC}}_{50} \;{\text{of}}\; M}} \times 100,$$
$${\text{Theoretical}}\;{\text{TI}}\;{\text{of}}\;M = {\text{TI}}\;{\text{of}}\;A \times \% \; {\text{of}}\;A\;{\text{in}}\;M + TI\;{\text{of}}\;B \times \% \; {\text{of}}\;B\;{\text{in}}\;M,$$
$${\text{Co-toxicity}}\; {\text{coefficient}} = \frac{{{\text{Actual}} \;{\text{TI}}\; {\text{of}}\;M}}{{{\text{Theoretical }}\;{\text{TI}}\;{\text{of}}\;M}} \times 100.$$
If one component of the mixture alone (for example a wood ash) causes low mortality at all doses (< 20%), then the co-toxicity coefficient of the mixture should be calculated by the formula:
$${\text{Co-toxicity}}\; {\text{coefficient}} = \frac{{{\text{LC}}_{50 } \;{\text{of}}\; A\; {\text{alone}}}}{{{\text{LC}}_{50 } \;{\text{of}}\; A\; {\text{in}}\; {\text{the}}\; {\text{mixture}}}} \times 100.$$
F1 progeny bioassay
After the 14-day mortality recordings, all insects and products were discarded. The grains were left inside the bottles, and the counting of F1 adults was carried out once a week during 5 weeks. The emergence started only from 5th week after infestation. After each counting session, the insects were removed from the jars [8].
Damage bioassay
Four rates of the binary combinations (5, 10, 20 and 40 g/kg) were mixed with 150 g of maize grain as described above. Fifty unidentified sex weevils (7–14 days old) were introduced into each jar. Each treatment had four replications. After 3 months, the live weevils and dead ones were counted. Damage assessment was performed by counting and weighing the number of damaged and undamaged grain using the method of Adams and Schulten [27].
$${\text{Weight}}\; {\text{loss}}\,\left( \% \right) = \frac{{\left( {W_{\text{u}} \times N_{\text{d}} } \right) - \left( {W_{\text{d}} \times N_{\text{u}} } \right)}}{{W_{\text{u}} \left( {N_{\text{d}} + N_{\text{u}} } \right)}} \times 100,$$
where Wu is the weight of undamaged grain, Nd the number of damaged grain, Wd the weight of damaged grain, and Nu the number of undamaged grain.
Test of germination
Seed germination was tested using 30 randomly picked grains from non-perforated grains after separation of the perforated from the non-perforated in each jar. In order to assess the effect of binary mixtures on germination ability, the seeds were treated with different contents and stored as previously described, but without insect. The seeds from the two lots (infested and non-infested) of stored maize grains were placed on moistened paper in 9-cm glass Petri dishes. The number of germinated seeds was recorded after 10 days [28].
Data analysis
Abbott’s formula [29] was used to correct for control mortality before analysis of variance (ANOVA) and probit analysis. Data on cumulative corrected mortality, reduction in F1 progeny, damage, weight loss and germination percentage were arcsine-transformed [(square root(x/100)], and the number of F1 progeny was log-transformed (x + 1). The transformed data were subjected to the ANOVA procedure using the statistical analysis system [30, 31]. Tukey’s test (P = 0.05) was applied for mean separation. Probit analysis [31, 32] was conducted to determine lethal dosages causing 50% (LC50) and 95% (LC95) mortality of S. zeamais at 1, 3, 7 and 14 days after treatment application. The probit analysis was also used to determine the effective content causing 50% (EC50) and 95% (EC95) reduction in F1 progeny.
