Musa A K， Odunayo A， and Adeyeye O E

Department of Crop Protection， University of Ilorin， Kwara State， Ilorin， Nigeria

Effects of Ιnitial Ιnfestation Levels of Callosobruchus maculatus （F.）（Coleoptera： Chrysomelidae） on Cowpea and Use of Nicotiana tabacum L. Aqueous Extract as Grain Protectant

Musa A K， Odunayo A， and Adeyeye O E

Department of Crop Protection， University of Ilorin， Kwara State， Ilorin， Nigeria

This study determined the effects of initial infestation of cowpea seeds （Ife brown variety） with different insect densities （0， 2， 4 and 6 pairs per 50 g seeds） of Callosobruchus maculatus （F.） and evaluated the effects of aqueous leaf extract of Nicotiana tabacum L. on C. maculatus in the laboratory. It was observed that adult beetle population increased significantly （p＜0.05）with increase in insect density. The increase in population of beetles and corresponding weight loss of the seeds in different levels of infestation showed that the cowpea variety was susceptible to beetle infestation， emergence and survival of progeny. Significantly more adults emerged on higher infestation compared to lower and no infestation. In Nigeria， Nicotiana tabacum L. is a locally available plant， with known insecticidal properties. The plant leaf extract was easily extracted with water and confirmed its effectiveness as a protective agent for stored cowpea seeds. Experiment was conducted to assess the effects of aqueous extracts of N. tabacum at 0， 0.1， 0.2 and 0.3 mL · 50 g-1cowpea seeds on C. maculatus. Data was recorded and showed varying levels of effectiveness against C. maculatus. Result showed that seed appearance was dependent on levels of insect population， while N. tabacum aqueous extract exerted effects on survival of C. maculatus. Aqueous leaf extract of N. tabacum probably contained some insecticidal properties which might have significantly conferred beetle mortality and reduced beetle emergence leading to a decrease in seed weight loss.

Callosobruchus maculatus， grain protectant， infestation， Nicotiana tabacum， seed damage

Ιntroduction

Cowpea， Vigna unguiculata （L.） Walpers （Papilionaceae）， is popularly grown in northern Nigeria with required climatic and soil conditions suitable for its cultivation. Its nutritional contents made it an important legume crop in human diet and livestock feeds. All the plant parts used for food are nutritious，providing protein， vitamins and minerals （Emeasor et al.， 2007）. The crop is， however， attacked by a number of insect pests both in the field and storage. Seed beetle， Callosobruchus maculatus （F.）， is a major field-to-store insect pest of cowpea responsible for a great deal of damage to the seeds making it unfit for human consumption. Musa （2007） estimated the loss caused by C. maculatus over a 12-week storage period to be 42.2% in Southern Guinea Savanna of Nigeria. Swella and Mushobozy （2009） examined the storability of 10 different legume seeds to infestation by C. maculatus over a period of 2 months and recorded mean weight losses ranging from 2.7 to 9.6 g.

Over the past few decades， application of synthetic，broad spectrum pesticides and fumigants to control stored product pest species had been a standard practice （Ukeh， 2009）. The insect pest has therefore，been controlled through the use of synthetic insecticides which include organophosphate and chlorinated hydrocarbons. However， these insecticides are not readily available to farmers because of their relatively high cost， pest resistance， lacking of application skills， adulteration， toxicity to man and animals （Afun et al.， 1991； Ajayi and Rahman， 2008； Musa et al.，2009）. The increasing problems of these insecticides to the environment have given credence to the desire for safer protectants， such as botanicals. Biopesticides are biodegradable and less harmful to man and environment （Schmutterer， 1990）， a result of which made them popular as potential pesticides with less challenges. The present study was undertaken to investigate insecticidal effects of aqueous extract of Nicotiana tabaccum against cowpea beetles，C. maculatus. The objective of the study was to determine the effects of different levels of C. maculatus infestation on cowpea and use aqueous leaf extract of N. tabacum as grain protectant.

Materials and Methods

Study area

The study was carried out in the Crop Protection Research Laboratory of University of Ilorin， Nigeria.

Source of cowpea seeds

The Ife brown variety of cowpea used for the study was obtained from the International Institute for Tropical Agriculture， Ibadan， Nigeria.

lnsect culture in laboratory

Callosobruchus maculatus used to establish the culture was obtained from a batch of already infested cowpea seeds kept in the Research Laboratory of the Department of Crop Protection， University of Ilorin，Ilorin， Nigeria. Fresh and clean cowpea seeds were purchased from a local market， Ilorin， and infested with beetles in a transparent plastic jar （9 cm diameter）covered with baft cloth to prevent insect escaping and entry of insects. The culture was maintained under ambient environment （28±3℃ and 75%±3% RH） of laboratory cupboards.

Preparation of plant material

Leaves of tobacco， Nicotiana tabacum， were plucked from the parent plant on an abandoned area， Sango，Ilorin. The leaves were washed and air-dried slowly in the laboratory for 21 days before pulverization in a mill. About 100 g of the plant powder were soaked in a closed vessel （30 cm diameter） of 1 L of water，stirred and left for 7 days. The solution was strained with a 0.1 mm sieve to standardize the particles，and solid residue was pressed followed by filtration through Whatman No. 1 filter paper. The filtrate was concentrated in a water-bath set at 40℃ to vapourize the excess solvent.

Bioassays

Effects of initial C. maculatus infestation on cowpea seeds

Freshly emerged adults of C. maculatus （about 2 days old） from the culture were introduced into 50 g clean cowpea seeds at 2， 4 and 6 insect pairs， respectively. The morphology of beetles' snout， that of the male was rough and coarse compared to that of the female which was smooth and uniform， was used for sex differentiation. The control was also set up with no insect in the experiment. Each mixture of seeds and beetles was covered in their containers with muslin to allow aeration and prevent beetle escaping or entry of predators. Data on population of surviving beetles and corresponding seed weight loss was taken at 30， 60 and 90 days after infestation （DAI）， while data on seed damaged was taken at 90 DAI.

Effects of aqueous leaf extract of N. tabacum on C. maculatus

Five teneral adults of three females and two males of C. maculatus were introduced to clean and wholesome cowpea seeds mixed with aqueous leaf extract of N. tabacum at the rates of 0.0， 0.10， 0.20 and 0.30 mL · 50 g-1seeds. The mixture was thoroughly agitated to achieve uniform coating of the seeds. Eachtransparent container was covered with muslin. Each treatment， including control （seeds not treated with leaf extract）， was replicated four times in a complete randomized design. Parameters quantified commenced with visual counts of dead adult beetles at 24 h intervals and was terminated at 96 h after treatment （HAT）. Keen observation on progeny emergence from 23 DAI was terminated at 35 days after treatment （DAT）， while the total emergence per treatment was used for computation. Extent of the cowpea seed damaged was determined 90 DAT under small-scale storage condition.

Statistical design

Data collected was subjected to one-way analysis of variance （ANOVA） and where significant differences existed， means were separated using Least Significant Difference （LSD） at p=0.05 significance level.

Results and Discussion

Table 1 showed the mean seed weight loss， number of seeds damaged as a result of feeding activities of C. maculatus and mean abundance of beetles. Seeds exposed to higher numbers of beetles had more damages and greater weight loss. It was observed that adult beetle population increased with increase in insect density. The increase in population of beetles in different levels of infestation showed that the cowpea variety was susceptible to beetle infestation， emergence and survival of the progeny. Mean population of adults increased significantly （p＜0.05） with increasing in initial levels of C. maculatus infestation. All the levels of infestation differed significantly （p＜0.05） in all the parameters observed from the control. The seeds in the control were not damaged at all as they were not infested. This study showed that C. maculatus readily attacked cowpea seeds exposed to infestation. The proximate compositions of cowpea made it possible for beetles to respond to the seeds. In this study，the seed weight loss， seed damaged and increase in beetle population were consequences of artificial infestation. Grain hardness had not been reported to confer complete immunity on grains from insect attack and infestation （Osipitan and Odebiyi， 2007）. However， there is need for further detailed studies on susceptibility of this variety to C. maculatus to determine inherent factors responsible for response of beetles to it. Idoko and Adesina （2012） observed that oviposition and adult emergence by C. maculatus differed significantly with insect density.

Table 1 Influence of different levels of Callosobruchus maculatus infestation on cowpea weight loss， seed damaged and insect abundance

The effects of rates of treatment of N. tabacum leaf extract on mean beetle mortality， progeny emergence of C. maculatus and seed weight loss and damaged by the insect are presented in Table 2. There was no adult mortality at 24 HAT except with the leaf extract applied at 0.3 mL which caused mean beetle mortality of 0.25. At 48 HAT， all rates of leaf extract caused mean mortality ranging from 1.0 to 1.25. The extract applied at 0.3 mL caused higher mean mortality of 4.50，96 HAT whereas the extract at 0.1 and 0.2 mL · 50 g-1cowpea seeds caused mean mortality of 3.50 and 4.25，respectively. Each rate of the leaf extract induced beetle mortality of C. maculatus significantly when compared with the control （Table 2）. Mortality varied with rates of treatments and exposure period as indicated by significant differences （p＜0.05） recorded among rates of treatments after a storage period of 96 HAT. Most beetles died with the highest rate of treatment and mortality increased by the day in all the treatments except in the control which had no mortality. The observed mortality might be linked to nicotine content of the leaf extract concentration and possibly vigour of beetles. The contact action of the extract might have inhibited respiration of adult beetles. Nicotiana tabacum was reported to possess contact， stomach and respiratory poisoning properties attributed to its active constituent， nicotine （Stoll， 1988）. The results confirmed the observation that mortality of insects varied with plant part， rate of application and exposure period （Akinneye and Ashamo， 2009）. The present study had shown that aqueous leaf extract of the plant materials could also be exploited for using as a grain protectant after careful consideration of nicotine which is a harmful component.

Table 2 Protective potential of aqueous leaf extract of Nicotiana tabacum against Callosobruchus maculatus in stored cowpea

Analyses of variance showed that there were significant differences in the mean number of emergence recorded at different rates of treatments. The higher rates of treatments of 0.3 mL gave the lowest adult emergence which was significantly different compared to the control. The highest rate of 3.0 mL was however comparable to other treatments except the control. The toxicity and potency of natural plants have been ascribed to their chemical active ingredients （Adedire and Lajide， 1999； Musa， 2007）.

Cowpea seeds treated with aqueous extract of N. tabacum suppressed adult emergence. Meanwhile，extract applied at 0.3 mL showed promising effects on suppressing adult emergence. Similar trend which was observed in adult emergence was also recorded for the mean weight loss. The mean weight loss on cowpea seeds increased with increase in initial number of C. maculatus population. The leaf extracts appliedat 0.2 mL · 50 g-1seeds and 0.3 mL · 50 g-1seeds gave a weight loss of 4.67 g and 2.26 g， respectively， which were significantly different compared to the control （10.96 g） at 60 DAT. The leaf extracts applied at 0.2 mL · 50 g-1seeds and 0.3 mL · 50 g-1seeds gave a weight loss of 5.96 g and 4.36 g， respectively which were significantly different compared to the control（13.29 g） at 90 DAT. The lowest level of treatment was not significantly （p＜0.05） different when compared to the control at 60 and 90 DAT. In this study， mean seed weight loss ranged from 2.26 in 0.3 mL to 8.29 in 0.1 mL at 60 DAT. Mean seed weight loss ranged from 4.36 in 0.3 mL to 8.90 in 0.1 mL at 90 DAT. The mean seed weight losses at 0.2 and 0.3 mL during the period of investigation were significantly different（p＜0.05） compared to the control. Aqueous extract applied at 0.3 mL was significantly effective in the control of C. maculatus at p=0.05 when compared to lower treatments. The performance of the leaf extract of N. tabacum showed when admixed at 0.1， 0.2 and 0.3 mL · 50 g-1cowpea seeds reduced adult emergence of C. maculatus by 28.85%， 46.63% and 68.27%，respectively compared to the control.

This experiment showed that N. tabacum might have some insecticidal properties which might have significantly reduced adult emergence from cowpea seeds， leading to a decrease in seed weight loss. Reduction in adult emergence could be ascribed to egg mortality or death of immature stages following the ovicidal and larvicidal properties of N. tabacum. Nicotine content might not afford recommendation of the plants for consumption of its toxicity also to humans through bonding to receptors at the nerve synapses （junctures）， causing uncontrolled nerve firing and by mimicking acetylcholine at the nervemuscle junctions in the central nervous system （Ujvary，1999）. Results showed that adult emergence and seed weight loss were insect population dependent and N. tabacum could be used for the effective management. Danjumma et al. （2009） reported that 100% adult mortality of Sitophilus zeamais was recorded on maize treated with the powder of N. tabacum. Khalif and Al-Farhani （2008） recorded that leaf powder of N. tabacum caused 100% mortality of Tribolium castaneum after 7 days.

Conclusions

It has been shown that N. tabacum leaf extract had some insecticidal effects against C. maculatus. Its inclusions in pest management strategies were therefore suggested for upgrading traditional postharvest protection practices of seeds required for consumption/animal feeding or next planting.

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Q969； S572 Document code： A Article lD： 1006-8104（2015）-04-0001-06

Received 1 August 2015

Musa A K， E-mail: akmusa2013@gmail.com