Laboratory evaluation of some botanicals and fermented cow urine against Chilo partellus (Swinhoe) (Lepidoptera: Crambidae)

Chilo partellus (Swinhoe) is the most important destructive pest of sorghum in Africa which results in complete crop loss. Currently the management strategies rely heavily on chemical insecticides, which do not provide effective control. The uses of bio-pesticides are encouraging over chemical pesticides. The study was carried out to identify effective plant and animal products and optimum concentrations against C. partellus. 7 treatments each at 3 concentrations (2.0, 2.5 and 3.0 g and 1.0, 1.5 and 2.0 ml) were applied on 2 nd and/or 3 rd instars larvae. The untreated check was used for comparison. The experiment was laid out in a completely randomized design with 3 replications for each concentration. Result revealed among the treatments Milletia ferruginea Hochst seed powder and aqua extract caused the highest (100%) mortality at 3 g and 2.0 ml concentration on the 2 nd day. Cow urine and the mixture (M. ferruginea+cow urine) were recorded higher efficacy mean mortality (86.7 and 93.3%) at 3.0 ml concentration within 3 days respectively. Phytolacca dodecandra L. seed powder, aqua extract and the mixture (P. dodecandra+cow urine) showed significantly lower efficacy mean mortality. In conclusion, M. ferruginea powder and aqua extract, cow urine and combination of M. ferruginea with cow urine were identified as good alternatives to chemical pesticides against C. partellus.


INTRODUCTION
Chilo partellus (Swinhoe) (Lepidoptera: Crambidae) is considered to be the most important pest of sorghum in sub-Saharan Africa including Ethiopia (Damte and Chichaybelu, 2002;Tesfaye and Gautam, 2003). Application of chemical insecticides has been recommended to protect sorghum from C. partellus attack.
However, chemicals are too expensive and are the cause of environmental and health hazards if not used judiciously (Gupta et al., 2010). Pesticide that have a sub-lethal toxicity to target pests, but still kill natural enemies of the pests may cause target pests to increase, resulting in even higher yield losses (Islam et al., 2013; *Corresponding author. E-mail: adem.nega1@gmail.com Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License  Khan et al., 2015). So these facts brought the need for search alternative pest control options, especially those that are cost effective and environmentally friendly. The use of plant and animal-based insecticides increases from time to time not only due to their availability but also their simplicity to prepare and increase quality of the crop in taste (Getu et al., 2008;Kareru et al., 2013). They are not phytotoxic, easy to grow, cause no hazard to non-target organisms, wild life, humans or the environment (Islam et al., 2013;Tilahun and Azerefegne, 2013). In most cases their bioactive compounds are fairly complex groups; thereby making it more difficult for the pest to develop resistance (Wahedi et al., 2016). However, there is no or less study carried out, or documented report regarding plant and animalbased insecticides efficacy test for managing C. partellus. Therefore, screening the effective plant and animalbased insecticides is crucial against this pest. Thus, this study was conducted to determine the efficacy of specific plant and animals-based insecticides and determine the best product that controls C. partellus.

MATERIALS AND METHODS
The study was conducted at Kombolcha Plant Health Clinic, Ethiopia, under ambient laboratory conditions in 2016/17. Eggs were collected from field 1 to 3 weeks after plants emergence. After 5 to 11 days, the newly hatched larvae were reared in a 600 ml rearing plastic beakers the top of which was covered with nylon mesh. Larvae were supplied pesticide free fresh sorghum leaves and stems. Seeds of M. ferruginea Birbira and P. dodecandra Endod were collected from Addis Ababa University, Arat-kilo Campus and road sides grown voluntarily plantations at Dessie town, Ethiopia. Cow urine was collected from the local dairy farm in the morning into a plastic container. Seeds were washed, dried and grounded into fine powder manually using home-made mortar and pestle (Jembere, 2002). The powder was kept separately in packed plastic bags in a refrigerator at 4°C until needed for its crude extractions. Each sample of 50 gm powder was weighed separately and mixed with 100 ml water in a separate flask for crude extractions (Venkat et al., 2012). The mixtures were stirred for 15 min using a magnetic stirrer until homogenous solutions was formed and then left to stand.
Each mixture was filtered through double folds Muslin cloth. After evaporating the solvent the solid extract was then used to prepare the serial dilutions. Each serial concentration was prepared by dissolving 100, 150 and 200 mg of each fine powder with 100 ml of distilled water (that is, to form 1, 1.5 and 2 ml concentrations) levels respectively. Cow urine was left 6+1 days in the shade at room temperature for fermentation; it was sieved and diluted with water at the ratio of 1:3 (v/v) (Verena, 2007). Treatment combinations were prepared by mixing equal volumes of cow urine with equal volume of each extracts. Treatments were applied on the 10, 2 nd and/or 3 rd instars larvae per Petri dish, and preserved food with pieces of sorghum stalk. Water was used as a control and all treatments were compared with the untreated check. All treatments each at 3 concentrations levels were laid out in a complete randomized design (CRD) with 3 replications for each concentration (Table 1). The numbers of larvae were recorded before spray and every 24 h after treatment applications till 3 days.

Data analysis
Data were subjected to analysis of variance (ANOVA) and significant means were separated by Least Significant Difference (LSD) at 0.05 level, using SAS program version 9.1 (SAS, 2009).

The effect of biopesticides on larval mortality of C. partellus after 1 day of treatment application
Effect of bio-pesticides on larvae of C. partellus showed that in all the rates, significant (P<0.05) differences were found among the treatments (Table 2). After 1-day treatment application, about 0.3 to 86.6% mortality was observed due to application of different bio-pesticide when compared to untreated control (0.3%). All treatments caused rate dependent mortality. The highest percent (86.6%) larval mortality was recorded on M. ferruginea aqua extract at the higher (2.0 ml) rate and the lowest percent (0.3%) mortality was observed at the lower (2 g and 1 ml) rates of P. dodecandra powder, aqua extract and cow urine + P. dodecandra respectively, however mortality at these treatments was not statistically different from the control.

The effect of biopesticides on larval mortality of C. partellus after 2 days of treatment application
Data on the mortality of the 2 nd and 3 rd instar larvae of C. partellus showed that all the treatments were highly significant (P<0.05) and superior over the control after 2 days treatment application (Table 3). All botanical treatments showed significantly (p< higher mortality) at the 2 nd days of exposure time compared to the control. The highest larval (100%) mortality was recorded on M. ferruginea powder and aqua extract at the higher (3 g) rate. Mortality at this rate was not statistically different from 2.5 g powder. The highest larval mortality recorded for M. ferruginea aqua extract was at 2.0 ml. P. dodecandra aqua extracts caused significantly lower (20.3%) mortality at the lower (1.0 ml) rate of application (Table 3).

The effect of biopesticides on larval mortality of C. partellus after 3 days of treatment application
Significant lethal effects of all the treatments on C. partellus larvae were found 3 days after treatment compared with the control. Mortality was increased as the rate of the extract applied increased in concentration. All botanical treatments showed significantly (p< higher mortality) at 3 days of exposure time compared to the control. The highest (100%) larval mortality was recorded on M. furrgunea powder and aqua extract at the higher rates. Mortality at the lower rates was not statistically different from the higher rates applications. The other treatments showed the highest larval mortality at the higher rates of applications. The lowest (26.6%) mortality was recorded in aqua extracts of P. dodecandra at the rate of 1.0 ml (Table 4).

DISCUSSION
Result of the laboratory experiment indicated that all treatments significantly resulted in C. partellus larval mortality compared to the untreated check. These results are in agreement with previous work of Shiberu et al. (2013) who reported that botanical products like water extracts of Birbira, Endode, Neem and Pyrethrum gave good control of Termites pests. There were high significant (P<0.01) difference among treatments in laboratory after exposure of 72 h. Among all the treatments used M. ferruginea powder and aqua extract were found to be the most toxic and caused the highest (86.67-100%) mortality within 48 h at the higher rate of applications. M. ferruginea seeds powder and aqua extract have been reported to have insecticidal properties. For example, M. ferruginea seed powder extracts resulted in a 96% mortality rate of maize weevils, Sitophillus zeamais 72 days after treatment application (Jembere, 2002).
The toxicity of the plant can be attributed to rotenone which is one of the dominant compounds found in the seed and stem bark of M. ferruginea and is a well-known botanical insecticide through contact and stomach poisoning (Gupta et al., 2010). Damte and Chichaybelu (2002) also tested the toxicity of Milletia seed against Adzuki bean beetle, Callsobruchus chinunesis and found that it gave complete protection of stored chickpea for six months in the laboratory.  14.5 14.5 14.5 LSD at 0.05 11.5 11.5 11.5 Means followed by upper letter across row and lower letter within column are not significantly different at 5% level, LSD.
Laboratory study on the toxicity of cow urine against C. partellus larvae caused high mortality (86.67%) as compared to the control. Similar result was reported in India by Tesfaye and Gautam (2003) who reported that cow urine caused 80% mortality of Welo bush cricket (WBC), Decticoides brevipennis (Raggea) in 12 h after treatment and reached 90% after 24 h and was at par with neem leaf extract. They also reported 79.6% Drosophila melanogaster (Meigen) mortality as compared with 2.8% in control and it was at least 3 times more effective than neem. Effect of the mixture (M. ferruginea aqua extract and fermented cow urine) was not significantly different from M. ferruginea powder and aqua extract (93.0%) at the higher rate of application, 2.0 ml after 72 h. Powder and aqua extracted of M. ferruginea caused significant larval mortality followed by the mixture (M. ferruginea aqua extract and fermented cow urine) at the lower rates. The other treatments show larval mortality at the higher rates of applications. This showed that from the different treatments used powder and aqua extracts of M. ferruginea found to be the most effective as compared to all other treatments.
High mortality due to M. ferruginea when compared to other plant products could be attributed to the presence of bioactive and other bitter compounds responsible for anti-feeding activity that result in the starvation and death of insects. Comparison of this result with previous work has shown consistency with Jembere et al. (2002) where water extracts of M. ferruginea caused higher toxicity to all the castes of termites in which 93 to 100% mortality was recorded at all concentration levels. M. ferruginea powder and seed kernel aqua extracts have been reported to be effective against various species of insects and are considered safe for human health and environments (Jembere, 2002;Muzeyi and Jembere, 2005;Taddese et al., 2010). In contrast, P. dodecandra aqua extract and the mixture (P. dodecandra and fermented cow urine) were least active as compared with the other treatments. Significant lethal effects of the treatments on larvae of C. partellus were found three days after treatment applications compared with the control. The assumption for this may be that the active compounds present in these treatments were less in amount. However, larval mortality due to these treatments was observed to increase as the exposure time of the pest to the treatment increased. As exposure time extends there was a progressive increase in the toxicity of these treatments to the test insect registering appreciable mortality of C. partellus larvae.
The present result clearly showed that P. dodecandra aqua extract and the mixture (P. dodecandra and cow urine) required three days to kill 53.3 and 60.0% of C. partellus. This implied mortality increases due to these treatments at the higher rates. Hence the days to higher larval mortality took significantly longer periods and required higher rates of applications. The probable assumption might be due to the slow acting effect of the potent insecticides present in these treatments. Comparison of this result with previous work has shown consistency to Shiberu (10) who reported that mortality of B. fusca increases at the concentrations of the treatments and exposure time of the insect increases.
Thus these treatments are less effective against C. partellus larvae compared to other treatments tested within three days, however they were considered as moderately effective treatments on C. partellus control. C. partellus is an internal feeder and when the larvae grow to be mature normally develops successfully inside the stem. Therefore, botanical extracts and fermented cow urine as a traditional pest control will completely increase the mortality rate of spotted stem borer, C. partellus when applied in the early stages of this pest.

Conclusion
The study concluded that many of the treatments tested appear to be quite effective as local source of insecticides. The efficacy of these treatments varied with Nega and Getu 5 different concentrations, time intervals and length of exposure time. However, all the treatments tested showed significant insecticidal property against C. partellus. Out of the 7 treatments tested, M. ferruginea powder and aqua extracts were having acute toxicity. P. dodecandra powder, cow urine and the mixture of M. ferruginea aqua extract with fermented cow urine were observed to relatively had high insecticidal activity. P. dodecandra aqua extract and its combination with cow urine were observed to have low insecticidal activity. Therefore, from the current study powder and aqua extract of M. ferruginea, cow urine and combination of M. ferruginea aqua extract with cow urine are identified as good alternatives to chemical pesticides in controlling C. partellus. However, further study is needed on this regard to confirm the efficacy of these plant and animal-based insecticides and their practical effectiveness under natural conditions against C. partellus without any side effects on none target organisms and the environment.