Sourcing of plant materials

The required quantity of the plant C. patens was completely uprooted from the source in the forest region along Akure-Ondo Road, Akure, Nigeria (located at latitude 7.2571°N and longitude 5.2058°E). The maize grains were bought at Isikan Market, Akure, Nigeria.

Preparation of plant materials

C. patens samples collected from the field were transported in protective bags to the Biology laboratory 2 of the Federal University of Technology, Akure.  The plants were thoroughly washed with water, then the root barks were carefully removed with sharp knife and air-dried in the laboratory for 50 days, after which they were pulverized into fine powder using Binatone electric blender (Model 373). The powdery samples were further sieved to pass through 1 mm² perforations to obtain labeled samples of fine powders that were kept in separate airtight plastic containers and stored at ambient temperature of 28±2°C and 75 ± 5% Rh pending use. The maize grains used were first sorted to remove both contaminants and damaged ones. The grains were later disinfected in the oven at 60°C for 4 h and allowed to cool on open laboratory bench for 5-6 h before been stored in plastic containers until usage. Who identified the botanical specimens of C. patens? and in which collections C. patens specimen vouchers were deposited?

Insect culture

500 g of maize grains was weighed into two kilner jars (1 L each). Ten newly emerged adults 5 males and 5 females P. interpunctella were introduced into each of the jars. The jars were kept in the culturing chamber until the F1 generation emerged. Insects were identified using standard entomological keys.

Contact bioassay of C. patens against stages of development of P. interpunctella

Twenty freshly laid eggs (0-24 h old) were placed on 20 g of maize grains treated with  0.5, 1.0, 1.5, 2.0, 2.5 g and control (untreated) leaf and stem bark powders were separately placed inside plastic container (8 cm diameter  and  4  cm  depth).  Each  treatment  was replicated thrice with a corresponding control. Daily observations were made with dissecting microscope to determine the number of hatching eggs from the total number of infested eggs. The experiments were randomly arranged and kept inside a breeding cage with wire mesh cage (75 × 50 × 60 cm). After the hatchability period (0-7days) the rearing containers were covered with muslin cloths and held in place with rubber bands; and during 40 days, the number and percentage of adult emerged was determined. The aforementioned procedure was repeated for both the larvae and adult contact bioassay. The same procedure was repeated for the larval and adult experiments; but the container covers were punched with hot iron rod and lined with muslin on the inside to prevent larval and adult from escaping and allow aeration. Ten larvae (third instar) were introduced into each bioassay treated and untreated grains and were replicated three times. The number of dead larvae and adult were counted and percentage mortality was determined. 

Fumigant bioassay of C. patens against stages of development of P. interpunctella

The following dosages; 0.5, 1.0, 1.5, 2.0 and 2.5 g and control (untreated) leaf and stem powders of the plant were separately weighed and sealed in muslin cloth (5cm by 5cm); they were hung on the lid of each of the plastic containers (8 cm depth 4 cm diameter). Twenty freshly laid eggs (0-24 h old) were introduced into each of the plastic containers containing 20 g of maize grains and covered with lid. The plant powder was hung between the lid and the bottom and was made air tight at equal distance. The treated and the control (untreated) were replicated three times. Daily observations were made using dissecting microscope to determine the number of eggs hatched from the total numbers of eggs introduced and the experiment was left inside the insect breeding wire mesh cage pending adult emergence. At the end of 40 days post-treatment period the total number of adults that emerged was determined and percentage mean was calculated. The same procedure was repeated for larvae. The dead larvae were counted and percentage mortality calculated after 24, 48, 72 and 96 h post treatment. The same procedure was repeated for the adult experiments.

The fumigant protocols are the same as the contact protocols, except that leaf and stem powders of the plant were separately weighed and sealed in muslin cloth (5 cm by 5 cm); they were hung on the lid of each of the plastic containers. The plant powder was hung between the lid and the bottom and was made air tight at equal distance. However, it is not clear whether the effect was caused by the release of gases from the powder or by particles from the powder itself. It is suggested to delete this part because C. patens was not actually used as a fumigant according to the protocols written in this study. Nevertheless, if the author or authors decide to keep it, then they have to do a one-way analysis of variance to compare Contact vs Fumigant toxicity.

Data analysis

Analysis of data was done using SPSS version 23. Means were separated using Turkey’s test.

Contact bioassay of C. patens against stages of development of P. interpunctella

The leaf  powder of C. patens was observed to be weakly effective against the spread of P. interpunctella when used as contact insecticide. Table 1 shows 63.33% egg hatchability at the highest dosage of 2.5 g powder. The same dosage achieved 69.17 and 45.83% larval and adult mortalities respectively after 96 h post-treatment exposure (Tables 2 and 3).

Unlike the leaf powder, C. patens stem powder was observed to be very effective in the control of the developmental stages of P. interpunctella. Table 4 reveals the contact effects of the powder on egg hatchability. It shows that hatchability was completely suppressed to 0% at 1.5 g powder dosage as against 63. 33% recorded when the leaf powder was used.  Table 5 reveals 100% larval mortality at 2.0 g powder dosage after 72 h post-treatment exposure. Also as contact insecticide, 100% adult  mortality  was  achieved  at 1.5 g dosage but after 96 h exposure as reflected in Table 6.

Fumigant bioassay of C. patens against stages of development of P. interpunctella

The leaf powder was slightly less effective in the control of the developmental stages of the pest when used as fumigant insecticide. Table 4 shows 68.33% egg hatchability at the highest powder dosage of 2.5 g. The same dosage yielded 36.26 and 53.33% larval and adult mortalities after 96 h post-treatment exposure (Tables 7 and 8). Tables 7 to 9 show that the fumigant insecticidal activities of the stem powder was observed to slightly less effective than the contact effect. Table 7 shows 0% hatchability  at   2.0 g   powder   dosage. It  was  also  0% under contact treatment but at 1.5 g dosage (Tables 10 to 12).

Food security for the increasing world population, most especially in the countries where pest control management is not of major concern had been and still being significantly challenged over years (Olotuah, 2014). Grain storage across the globe had been relying so heavily on the use of synthetic pesticides against insect infestation, the use of which have triggered a number of ecologica, health-related and pest resistance problems (Verma and Derbey, 1999). Works on organic pesticides to make up for these short falls had been promising. Several  botanical   products   have   been  discovered as potent in the control of storage pest infestation (Ofuya and Dawodu, 2002; Adedire and Ajayi, 2003; Tan and Luo, 2011). The results of this study have shown that the botanical powders of various compositions from C. patens are toxic to egg, larval and adult stages of P. interpunctella in stored products, most especially maize grains. This is in agreement with Akinneye et al. (2006) that showed the efficacy of root bark, stem bark and leaf powders of C. patens at varied compositions both as contact and fumigant insecticides in the control of egg and adult emergence stages of some Coleopteran and Lepidopteran storage pests. This result reveals a significant contact effect as compared with the fumigant effects of the powders on the moth pest. A concentration of 1.5 g leaf powder at all levels affected 100% egg, larval and adult mortalities after 72 h exposure when used  as  contact  insecticides. The  fumigant  effect  only achieved 23.37, 21.67 and 75.56% egg, larval and adult mortalities respectively at the same concentration. The fumigant  effect   of  2.0 g  concentration  achieved  100% larval and adult mortalities after 96 h exposure.

The inability of the eggs to hatch may be because powder inhibits gaseous exchange between the eggs and their external environment (Akinneye, 2003). The relatively high mortality rate of C.  patens to the pest may be attributed to the chemical composition of the powder just as reported by Ketunku et al. (2014) that saponin found in Eugenia aromatic (L.)[Bail] affected the respiratory system of certain storage insects thereby preventing their spread. It may also be attributed to the odour or characteristic bitterness associated with the leaf powder.

This corroborates the findings of Lale and Abdurahman (1990) that mortality of storage insects could be associated with pungent odour produced by plant powders against them. The finding is also in line with the report of Akinneye (2003) that C.patens inhibits egg hathchability and development of adult stages of Ephestia cautella (Walker)[Lepidoptera; Pyralidae]. Ashamo and Ogungbite (2014) also discovered that  E.aromatica prevented the emergence of some adult storage moths even at 2% concentration. This result is also in agreement with the work of Adedire and Lajide (2001), and Longe (2004) that E.aromatica powder has significant contact and fumigant actions on Calosobruchus maculatus (F.) (Coleoptera: Bruchidae). The progressive reduction in percentage adult emergence with increasing concentration and exposure period could suggest the death of the pests at larval stage due to their inability to fully cast off their exoskeleton which remains a link to the posterior parts of their abdomen just as reported by Oigiangbe et al. (2010). This result also tallies with the findings of  Adedire  and  Lajide  (2001)  that  spulverized powder of Piper umbrellatum (Linn.)[Piperaceae] seed and E. aromatica were toxic to C. maculatus, producing 100% mortality at 24 h post treatment across all concentrations. This study had revealed positive contact and fumigant effects of Cleitopholis patens powders on the P. interpunctella across the concentration gradient and exposure period. The findings suggest that the botanical product could serve as an alternative to synthetic chemicals against P. interpunctella.

The authors have not declared any conflict of interests.